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with, epitope, that, from, mass, were, antibodies, Figure, antibody, peptide, peptides, sequence, identification, using, cleavage, discussion, binding, Results, amino, proteolytic, Aß(1-40), antigen, amyloid, which, affinity, spectrometric, fragments, residues, acid, been

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Molecular Identification of Antigen Recognition Structures in
Immune Complexes for Immunotherapeutic Applications by
Proteolytic and Mass Spectrometric Methods
Dissertation
zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften
an der Universität Konstanz
vorgelegt von
Raluca Stefanescu
Konstanz 2007
Tag der műndlichen Prűfung: Donnerstag, den 13. Dezember 2007
Vorsitzender und műndlicher Prűfer: Herr Professor Dr. Fischer
Műndliche Prűfer: Herr Professor Dr. Dr. h.c. Przybylski
Herr Professor Dr. Jeschke
It is not the possession of truth, but the success which attends the seeking after it,
that enriches the seeker and brings happiness to him.
Max Planck
For my wonderful parents Angela and Radu Stefanescu
The present work has been performed in the time from September 2003 to June
2007 in the Laboratory of Analytical Chemistry and Biopolymer Structure Analysis,
Department of Chemistry of the University of Konstanz, under the supervision of
Prof. Dr. Dr. h. c. Michael Przybylski.
Special thanks to:
Prof. Dr. Dr. h. c. Michael Przybylski for giving me the opportunity to work in his
group, for the interesting research topic and discussions concerning my work and
for his entire support;
Prof. Dr. Gunnar Jeschke for writing the second evaluation of my dissertation;
Prof. Dr. Sylvie Rebuffat and Dr. Severine Zirah for the collaboration at the Zn2+
effect on the antibody interaction to amyloid peptides.
Prof. Dr. Michael Ehrmann for providing the recombinant HtrA and C99.
Prof. Dr. Richard Dodel and Dr. Michael Bacher for providing human Aß-antibody
samples (IVIG, AD77, 105, 005, 006) employed in epitope identification.
Prof. Dr. Beat Ernst and Dr. Rita Born for providing the antibody and the
recombinant H1CRD employed in this work.
All members of the group for the nice and inspiring atmosphere, but most of all I
want to thank to Dr. Xiaodan Tian, for her didactic attitude at the beginning of the
work in the research group, Dr. Marilena Manea, Dr. Eugen Damoc, Dr. Roxana
Iacob, Dr. Andreas Marquardt, Dr. Catalina Damoc, Iuliana Susnea, Alina Petre,
Dr. Suzanne Becker, Madalina Maftei, Adrian Moise, for scientific discussions and
interesting advices during my work; Claudia Cozma, Camelia Vlad and Stefan
Slamnoiu for the dedicated work during the practicals; Lana Mack, Gabriela
Paraschiv and Ute Schad for all the help they gave me, Reinhold Weber for the
organization and expert group leading in the Swiss Alps.
Special thanks also to my friends: Marilena, Eduard, Ana-Maria, Lacramioara,
Livia and Ioana for their support and the wonderful time spent together.
Last but not least I wish to thank and to express my deep gratitude to my family,
for supporting and encouraging me during this time.
This dissertation has been published in part, and presented at the following
conferences:
Peer-Reviewed Publications
1. Zirah S., Stefanescu R., Manea M., Tian X., Cecal R., Kozin D.A., Debey P.,
Rebuffat S., Przybylski M. (2004) “Zinc binding agonist effect on the recognition of
the ß-amyloid (4-10) epitope by anti-ß-amyloid antibodies”, Biochem. Biophys. Res.
Commun. 321(2), 324-328.
2. Grau S., Baldi A., Bussani R., Tian X., Stefanescu R., Przybylski M, Richards P.,
Jones S.A., Shridhar V., Clausen T., Ehrmann M. (2005) “Implications of the
serine protease HtrA1 in amyloid precursor protein processing”, Proc. Natl. Acad.
Sci. 102(17),6021-6026.
3. Tian X., Cecal R., McLaurin J., Manea M., Stefanescu R., Grau S., Harnasch M.,
Amir S., Ehrmann M., St. George-Hyslop P., Kohlmann M., Przybylski M. (2005)
"Identification and structural characterisation of carboxy-terminal polypeptides and
antibody epitopes of Alzheimer's amyloid precursor protein using high resolution
mass spectrometry", Eur. J. Mass Spectrom. 11(5), 547-556.
4. Bilkova Z., Stefanescu R., Cecal R., Korecka L., Ouzka S., Jezova J., Viovy J.L.,
Przybylski M. (2005) “Epitope extraction technique using a proteolytic magnetic
reactor combined with Fourier-transform ion cyclotron resonance mass
spectrometry as a tool for the screening of potential vaccine lead peptides”, Eur. J.
Mass Spectrom. 11(5), 489-495.
5. Stefanescu R., Iacob R., Damoc N., Marquardt A., Amstalden E., Manea M.,
Perdivara I., Maftei M., Paraschiv G, Przybylski M., (2007) “Mass spectrometric
approaches for elucidation of antigen-antibody recognition structures in molecular
immunology”, Eur. J. Mass Spectrom, 13(1), 69-75.
Conference presentations
1. Stefanescu R., Cecal R., McLaurin J., Tian X., Manea M., St. George-Hyslop P.,
Przybylski M., (2003) “Mass spectrometric and immunoanalytical characterisation
of an amyloid plaque-specific epitope of a therapeutically active anti-Aß42
antibody from transgenic mice”, DGMS, Münster, Germany.
2. Stefanescu R., Tian X., Grau S., Ehrmann M., Przybylski M., (2004)
“Characterisation of the Proteolytic Reactivity and Specificity of Enzymes on
Alzheimer-Target Proteins by High Resolution Mass Spectrometry”, 52nd
Conference of Mass Spectrometry and Allied Topics, Nashville, Tennessee, USA,
3. Stefanescu R., Tian X., Grau S., Ehrmann M., Przybylski M., (2005) “High
resolution mass spectrometric cleavage site elucidation of the novel serine
protease HtrA involved in amyloid precursor protein processing”, Human Proteome
Organisation (HUPO) 4th Annual World Congress, München, Germany.
4. Stefanescu R., Born R., Slamnoiu S., Ernst B., Przybylski M., (2006) “Mass
spectrometric and immunoanalytical epitope characterisation of a specific antibody
to the H1-carbohydrate recognition domain of the asialoglycoprotein receptor”, 17th
International Mass Spectrometry Conference, Prague, Czech Republik.
5. Stefanescu R., Iacob R., Manea M., Tian X., Perdivara I., Maftei M., Paraschiv G.,
McLaurin J., St. George-Hyslop P., Przybylski M., (2007) “Epitope identification
and structure determination of Aß-specific antibodies upon Aß-Immunisation using
High-Resolution Mass Spectrometry”, 8th International Conference AD/PD,
Salzburg, Austria.
I
TABLE OF CONTENTS
1. INTRODUCTION ........................................................................................ 1
1.1. Biochemical and immunological basis of antigen-antibody recognition ...... 1
1.1.1. Immune response: generation of antibody diversity ................................... 1
1.1.2. Structure of antibodies and recognition of antigens .................................... 3
1.2. Analytical methods for epitope identification ............................................... 6
1.3. Identification of epitopes by proteolytic cleavage and mass
spectrometry ............................................................................................... 7
1.4. Pathophysiological characteristics and therapeutic perspectives of
Alzheimer´s Disease ................................................................................. 13
1.5. Immunotherapeutic strategies for Alzheimer´s Disease ........................... 17
1.6. Scientific goals of the dissertation ............................................................ 21
2. RESULTS AND DISCUSSION ................................................................. 23
2.1. Characterisation of Aß-plaque specific antibodies .................................... 23
2.1.1. Metodology of mass spectrometric epitope identification ......................... 23
2.1.2. Mass spectrometric elucidation of the epitope recognized by
polyclonal anti-Aß42 and monoclonal anti-Aß(1-17) antibodies ............... 25
2.1.3. Identification of functional amino acid residues within the ß-amyloid
plaque specific epitope using alanine-scanning mutagenesis .................. 28
2.1.4. Effect of zinc ions on the recognition of N-terminal ß-amyloid epitope
by plaque specific antibodies .................................................................... 34
2.2. Epitope elucidation of Aß-specific human antibodies ............................... 42
2.2.1. Therapeutic potential of amyloid-specific autoantibodies from human
serum ................................................................................................. 42
2.2.2. Purification of Aß-autoantibodies from pooled immunoglobulin
gamma preparations ................................................................................. 44
2.2.3. Identification of the epitope recognized by Aß-autoantibodies isolated
from pooled immunoglobulin gamma ........................................................ 46
2.2.4. Clonal diversity and sequence analysis of human serum Aß
antibodies by 2D-gel electrophoresis and peptide mass fingerprint .......... 56
2.2.5. Identification of the epitope recognized by Aß-autoantibodies isolated
from the serum of an Alzheimer disease patient ....................................... 63
2.2.6. Synthesis of biotinylated amyloid peptides encompassing the epitope .... 65
2.2.7. Characterisation of the human Aß-autoantibodies binding to amyloid
peptides by ELISA .................................................................................... 67
II
2.2.8. Concluding discussion of the Aß-autoantibody epitope ............................ 72
2.3. Investigation of the cleavage specificity of ß-amyloid peptides by
HtrA1 protease ......................................................................................... 74
2.3.1. Structure and biological functions of HtrA1 ............................................... 74
2.3.2. Clearance of cerebral Aß by enzymes ...................................................... 75
2.3.3. Analytical characterization of C99 and HtrA ............................................. 76
2.3.4. Mass spectrometric identification of cleavage sites in C99 and Aß .......... 77
2.4. Epitope identification of a monoclonal antibody to the H1carbohydrate recognition domain (H1CRD) of the asialoglycoprotein
receptor .................................................................................................... 83
2.4.1. Structure and biological functions of H1CRD ........................................... 83
2.4.2. Primary structure characterisation of H1CRD using mass
spectrometric methods ............................................................................. 85
2.4.3. Epitope identification of a monoclonal antibody to H1CRD ...................... 96
2.4.4. Affinity of synthetic epitope peptides to the monoclonal antibody ........... 100
3. EXPERIMENTAL PART ......................................................................... 103
3.1. Proteins, Enzymes and Antibodies ......................................................... 103
3.2. Materials and reagents ........................................................................... 103
3.3. Solid phase peptide synthesis ................................................................ 104
3.4. Chromatographic and electrophoretic separation methods .................... 106
3.4.1. Reversed phase-high performance liquid chromatography .................... 106
3.4.2. Sample concentration and desalting using Zip Tip pipette tip ................. 108
3.4.3. One-dimensional gel electrophoresis ..................................................... 108
3.4.3.1. SDS-PAGE according to Laemmli .......................................................... 109
3.4.3.2. SDS-PAGE according to Schägger and Jagow ...................................... 110
3.4.4. Two-dimensional gel electrophoresis ..................................................... 111
3.4.5. Colloidal Coomassie staining.................................................................. 112
3.4.6. Silver staining ......................................................................................... 112
3.5. Immunological methods .......................................................................... 113
3.5.1. Preparation of immobilised antibodies .................................................... 113
3.5.2. Epitope excision and extraction experiments.......................................... 113
3.5.3. Preparation of immobilized antigen column ............................................ 114
3.5.4. Separation of Aß-autoantibodies from pooled IgG preparations ............. 114
3.5.5. Enzyme-linked immunosorbent assay .................................................... 115
3.5.5.1. Alanine scanning mutagenesis ............................................................... 115
3.5.5.2. Zinc binding effects on the antigen-antibody interaction ......................... 117
III
3.6. Chemical modification reactions and enzymatic fragmentation of
proteins ................................................................................................... 119
3.6.1. Reduction and alkylation of disulfide bonds in solution ........................... 119
3.6.2. Proteolytic digestion of proteins in solution using trypsin ........................ 119
3.6.3. Proteolytic digestion of proteins in solution using endoproteinase
GluC ................................................................................................... 119
3.6.4. Proteolytic digestion of proteins in solution using alpha-chymotrypsin ... 120
3.6.5. Proteolytic digestion of proteins in solution using Pronase ..................... 121
3.6.6. Proteolytic digestion of proteins in solution using Endoproteinase
LysC ................................................................................................... 121
3.6.7. In-gel trypsin digestion procedure of Coomassie Brilliant Blue stained
proteins ................................................................................................... 122
3.7. Mass spectrometric methods .................................................................. 122
3.7.1. Time of flight mass spectrometry ............................................................ 122
3.7.2. Fourier-transform Ion-Cyclotron Resonance mass spectrometry ........... 124
3.7.2.1. MALDI-FT-ICR mass spectrometry ......................................................... 125
3.7.2.2. ESI-FT-ICR mass spectrometry .............................................................. 126
3.7.3. Liquid chromatographic/Ion trap mass spectrometric investigation ........ 127
3.8. Bioinformatic tools for mass spectrometry .............................................. 128
3.8.1. GPMAW ................................................................................................. 128
3.8.2. Search engines for identifying proteins ................................................... 129
3.8.3. BALLView 1.1.1 ...................................................................................... 129
3.8.4. PDQuest 2-D gel analysis software ........................................................ 130
4. SUMMARY ............................................................................................. 131
5. ZUSAMMENFASSUNG ......................................................................... 135
6. REFERENCE LIST ................................................................................. 139
7. APPENDIX ............................................................................................. 156
7.1. Appendix 1 ............................................................................................. 156
7.2. Appendix 2 ............................................................................................. 158
7.3. Appendix 3 ............................................................................................. 159
7.4. Appendix 4 ............................................................................................. 163
Introduction 1
1. INTRODUCTION
1.1. Biochemical and immunological basis of antigen-antibody recognition
1.1.1. Immune response: generation of antibody diversity
The immune response is activated by the presence of foreign microorganisms that
elude the anatomic and physiologic host barriers, and by genetically modified own
cells that express unknown proteins. The two branches of the immune responses, i.e.
humoral and cell-mediated, assume different, complementary roles in protecting the
host [1-3].
The humoral response is responsible for the neutralization of extracellular invaders
and soluble foreign molecules and is mediated by B-cells. Each of the mature B-cells
expresses about 105 identical membrane bound antibody molecules (IgM and IgD)
with a unique amino acid sequence within the population of B-cells. During B-cell
maturation in the bone marrow an enormous number (1010) of antigen-specific
membrane antibodies can be generated despite the reduced repertoire of
immunoglobulin genes [4-7]. This enormous diversity is the result of random gene
rearrangements and is later reduced by a selection process that eliminates any B-cell
with membrane-bound antibody that recognizes self components. The first encounter
with a foreign entity capable of binding to the membrane antibody leads to the
stimulation of the B-cell which divides rapidly generating memory B cells and effector
B cells (see Figure 1). Memory B cells express the same antibody bound to the
membrane and are able to generate new memory cells and effector cells.
Mature B-cell
Expressing unique
antibody molecules
Antigen
stimulation
Activated B-cell
Division and
differentiation IgM
IgG
Class
switching
Memory B-cell
Effector B-cell
Effector B-cell
Figure 1: Overview of the humoral immune response. After the interaction with an antigen, B
cells divide and differentiate into memory B cells and effector B cells. The latter
produce soluble antibodies able to interact specifically to the antigen.
Introduction 2
Punctual mutations in the recognition site of the antibody which occur in a random
manner, followed by the preferential selection of these memory B cells with highest
affinity for the antigen increase the binding efficiency of the population of B cells. The
effector B cells, called also plasma cells, secrete soluble antibody molecules which
bind to the antigen facilitating its clearance from the body.
In contrast to the humoral immunity which provides protection against extracellular
invaders, the cell-mediated immunity is able to detect and eliminate cells that harbor
intracellular pathogens and cancerous cells that express non-self molecules. In the
cell mediated immune response the antigen is recognized by T cells which carry
approximately 105 membrane-bound T-cell receptors whose structural diversity
resembles those of the B cell receptors. However, the T cell receptor does not exist
in soluble form and recognizes the antigen only if is enzymatically degraded and the
peptide fragments are presented by the major histocompatibility complex (MHC)
molecules (see Figure 2). Therefore, the T cell epitopes have to be considered as
part of a molecular complex formed by the antigen presenting molecules and antigen
fragments.
Figure 2: Processing and presentation
pathway of the exogenous antigens. The
foreign agent is degraded by antigen
presenting cells and the peptide fragments
resulted are associated with class II MHC
molecules and exposed at the surface of the
cell. The peptide-MHC complex is
recognized by a T-helper cell which binds to
the complex.
T helper cell
Antigen presenting cell
CD4
Antigen
Class II MHC-peptide complex
T cell receptor
Introduction 3
An exogenous antigen, internalized by the antigen-presenting cells (macrophages,
dendritic cells and B cells) by endocytosis is degraded into peptides while moving
through several acidic, enzyme containing compartments, and is finally associated
with class II MHC (major histocompatibility complex) molecules transported in
vesicles from the Golgi complex. The class II MHC-peptide complex is ultimately
transported to the plasma membrane and exposed at the surface of the cell (see
Figure 2). The peptides combined with MHC class II are recognized by T helper cells
displaying the coreceptor CD4. Virus-infected host cells and cancerous cells express
endogenous antigenic proteins which are degraded within the endoplasmic reticulum
into peptide fragments. After binding to class I MHC molecules the complex is
transported to the cell membrane where it is recognized by T-cytotoxic cells
displaying the coreceptor CD8.
1.1.2. Structure of antibodies and recognition of antigens
Antibodies are antigen-binding proteins present on mature B-cell membrane
(monomeric IgM and IgD), and are secreted by the effector B cells as IgM, IgG, IgA,
IgE. The secreted immunoglobulins each have identical amino acid sequences within
the antigen binding site with the membrane bound immunoglobulin of the B-cell clone
activated by the interaction with the antigen, but differ in the constant region as result
of the class switch process (see Figure 1) [5]. Although IgG is the most abundant
class in serum (about 80 % of the total immunoglobulin), each class has specific
structural and functional properties.
As depicted in Figure 3a, immunoglobulin G consists of two types of polypeptide
chains, a 25-kDa light (L) chain and a 50-kDa heavy (H) chain. Each heavy chain is
linked to a light chain by a cystinyl-bond, and the two heavy chains are bound to
each other by disulfide bridges and non-covalent interactions. The complete
molecule adopts a conformation that resembles the letter Y. The light and heavy
chains are folded in 2 and 4 globular domains comprising approximately 110 amino
acids.
Introduction 4
a) b)
The immunoglobulin domain consists of a pair of ß-sheets each built of antiparallel ß
strands linked by a single disulfide bridge. The amino-terminal immunoglobulin
domain on each of the light and heavy chain termed variable domain displays a
higher variation of the amino acid sequence and contains three connecting loops at
one end of the ß-sheet characterized by a hyper variability of the amino acids. The
hypervariable loops are referred to as complementarity determining regions (CDRs)
[8]. The CDRs of the variable domains from each pair of heavy and light chain, form
a single surface located at the end of each arm which interacts with the antigen. The
amino acid residues involved in the interaction with the antigen form the paratope [9].
The remaining immunoglobulin domains on each heavy and light chain are similar in
all antibodies and are referred to as constant domains. A ribbon representation of the
crystal structure of the non-covalent complex formed by the hen eggwhite lysozyme
(HEL) and the Fab of the HyHEL5 antibody is shown in Figure 3b [10, 11].
Figure 3: Schematic representation of the immunoglobulin G and the antigen binding site. a) The
heavy chain consists of 3 constant domains (CH) and a variable domain (CV) and is
linked through a disulfide bridge to a light chain composed of a constant (CL) and a
variable (VL) domain. Two heterodimers made of a heavy and a light chain are linked to
form the IgG molecule. The CDRs are depicted according to the Kabat numbering [10];
b) Ribbon diagram from the crystal structure of the anti-HEL Fab shown in complex
with the lysozyme. The hypervariable regions (CDRs) (yellow) located on the variable
domains of the heavy (red) and light (blue) chain form the binding cleft which interacts
with the antigen (Protein Data Bank [9] accession number 1YQV).
Antigen
NH3+
-S
-S
- -S-S-
-S-S-
-S-S-
NH3+
COO- COO-
COO-
Fc VL
CL
VH
CH1
CH2
CH3
COO-
Antigen binding
domain
Fab
31-35
50-65
95-102
24-34
50-56
89-97
VH
CH1
VL
CL
CDRs
Introduction 5
Antibodies recognize discrete sequences of amino acids located at the surface of the
antigen called epitopes or antigenic determinants. To form an immune complex, noncovalent interactions (hydrogen bonds, ionic bonds, hydrophobic interactions, Van
der Waals interactions) are established between the surfaces of the 2 molecules,
complementary in their topology [12]. Approximately 90 % of the antibodies raised
against intact proteins recognize a discontinuous epitope (see Figure 4) consisting of
amino acid residues localized on different regions of the linear amino acid sequence
of the antigen, that are folded together in the native molecule. Denaturation of the
protein leads in most cases to the loss of antibody affinity. Only 10 % of the
antibodies are estimated to interact with continuous epitopes that are composed of
contiguous amino acid residues along the polypeptide chain [13].
Proteins with more complex structures have usually multiple antigenic determinants.
The x-ray crystallographic analyses of the immune complexes of 3 antibodies
HyHEL-10, HyHEL-5 and D1.3 raised against the globular antigen hen egg-white
lyzozyme revealed that all recognize discontinuous epitopes. While the HyHEL-5 and
D1.3 interact with distinct epitopes consisting of 2 streches of polypeptide chains, the
sites that interact with HyHEL-10 are more dispersed within the primary structure of
the lyzozyme [14, 15].
The ability to induce a humoral immune response is called immunogenicity and
depends on the capacity of the immune system to distinguish between self and
nonself agents, molecular size of a foreign agent, its chemical composition and
heterogeneity. In contrast to the immunogens, antigens are molecules that interact
specifically with the immunoglobulins without having the ability to elicit an immune
H2N
COOH
continous
epitope
discontinous
epitope
Discontinuous
epitope
Continuous
epitope
Figure 4: Schematic
representation of a
continuous and a
discontinuous epitope.
Introduction 6
response. Upon immune challenge by a foreign agent, different B-cell clones, each
with unique amino acid sequence and conformation of the combining site, are able to
interact with the immunogen. Each clone interacts with a different sequence of amino
acids on the surface of the immunogen and will secrete a specific antibody for this
sequence. The affinity separation of the IgG molecules interacting with the
immunogen produces a purified polyclonal antibody. In contrast, by fusion of an
activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell) a
hybrid cell is formed, called a hybridoma, that posseses the immortal-growth
properties of the myeloma cell and secretes only the antibody produced by the
activated B cell. The affinity separation of the IgG molecules using the immobilized
immunogen will lead to a purified monoclonal antibody.
1.2. Analytical methods for epitope identification
In the past decade a variety of methods, including mass spectrometry-based
approaches have been developed to provide structural information about antigenantibody complexes. In particular, the elucidation of the fine structure of the epitopes
provides a basis for the design of diagnostic tools and improved immunogens as lead
structures in the development of vaccines [16-18]
One approach to the identification of the epitope is termed Pepscan and consists of
the synthesis of overlapping peptide sequences that will be further scanned for the
binding to the specific antibody by enzyme-linked immunosorbent assay (ELISA) [1922]. There are different strategies used for the synthesis of the peptides. The
peptides can be synthesized in larger amounts on a resin, cleaved from the support
and purified for the binding assay, or they can be synthesized directly on the surface
on which the binding assay takes place (multi-pin synthesis) [23]. The first approach
allows the use of fresh peptides for each test while the second implies the use of
regenerated peptides after dissociation of the immune complex. The use of synthetic
peptides for testing the reactivity to the antibody allows also the replacement of
specific amino acids present in the antigen sequence with alanine providing the
identification of critical amino acids (alanine scanning mutagenesis) [24, 25].
Introduction 7
Alternatively, proteins containing alanine mutated amino acids are produced by
expression [26, 27].
Phage display has been also successfully used for the identification of linear epitopes
[28, 29]. The method is based on the incorporation of a nucleotide sequence
encoding a foreign peptide into a phage genome as a fusion to a gene encoding a
phage coat protein. This fusion ensures the display of the foreign peptide. Selection
of the phage displaying the interacting peptide is achieved by exposing the phage
particles to an immobilized antibody, removal of non-binding and non-specifically
bound phages by several washes and recovery of the bound phages by acid elution.
The interacting peptide sequence is identified by sequencing the phage genome
which contains the fusion gene coding the peptide.
To date X-ray crystallography is the most powerful technique for structure
determination and analysis of proteins because it is capable of providing atomic
coordinates of an entire assembly [30]. However, a critical step is the crystallization
of the sample which requires large amounts of protein with high purity for screening a
wide range of conditions (pH, temperature, salt, protein concentration) to find the
appropriate conditions. In contrast NMR spectroscopy experiments require
solubilisation of the protein in aqueous buffer under conditions similar to the
physiological conditions. The major drawbacks of the method are the time consuming
data collection and data analysis, the size constraints of the proteins and amount of
sample required for analysis.
1.3. Identification of epitopes by proteolytic cleavage and mass
spectrometry
Mass spectrometry has emerged as a widespread technique for the study of protein
structure, function, quantity and interaction with other biomolecules. Important
features of the mass spectrometric protein analysis are the high sensitivity, high
mass accuracy, short analysis time and low sample consumption. To identify
molecules within complex protein mixtures and to dissect the structure of the
molecular recognition domains diverse applications have been developed in
Introduction 8
conjunction with mass spectrometry. These methods include chromatographic and
electrophoretic separations, proteolytic assays, and differential chemical modification
of specific amino acid functions, sample preparation and bioinformatic tools for data
analysis.
One of the most important applications of MS is that it provides structural
identification of epitopes, unlike any of the other methods. The first attempts
concerning the investigation of the antigenic determinant resulted by limited
proteolytic cleavage of immune complexes were carried out by PAGE [31] and HPLC
[32]. However both methods are unsuitable for unambiguous epitope identification. A
general, molecular approach for identification of epitopes from peptide and protein
antigens using mass spectrometry was developed by our laboratory [33, 34]. This
method combines the advantage of the proteolytic stability of antibodies, and the
shielding of the epitope with the unambiguous molecular identification provided by
MS [16, 35, 36].
For epitope excision, the antigen is digested by proteolytic enzymes following the
formation of the immune complex (see Figure 5). The epitope will be resistant to the
fragmentation while the peptide sequences exposed to the enzymes will be cleaved.
Cleavage sites of the protease located inside the antigenic determinant that are not
fragmented provide information on the epitope. The peptide fragments resulting after
cleavage by the proteolytic enzyme as well as the epitope fragments collected after
acidic dissociation of the complex are analyzed by mass spectrometry which is
capable to provide unambiguous identification of the peptide sequences. In epitope
extraction, the proteolytic fragmentation of the antigen in solution yields peptide
sequences that might contain the intact epitope if the enzyme has no specificity for
the amino acids contained by the antigenic determinant. The resulting peptide
fragments are allowed to react with the antibody. Due to the high specificity of the
antigen-antibody interaction only the peptides that contain the antigenic determinant
in a similar conformation as in the intact protein will interact. The characterization of
discontinuous epitopes is usually more difficult involving a combination of proteolytic
epitope excision, chemical modification and mass spectrometry [37-41].
Introduction 9
a) b)
The methodology for epitope excision using proteolytic cleavage of the unbound
peptide sequences followed by mass spectrometric identification of the remaining
affinity bound peptides can be employed for the identification of the binding partners
of any isolated protein, and therefore, emerged as an important tool in the
characterization of protein function [42]. The identification of the specific antigen from
a complex mixture of proteins is achieved based on the data base search using the
peptide masses determined from the elution fraction. An immobilized anti-troponin
antibody and bovine heart cell lysate were used as model system.
A new, recent development, as an analogous method to the determination of
epitopes has been the identification of antibody paratope structures using antigen
columns containing the immobilized epitope. The antibody is exposed to the antigen
column either after digestion in solution (proteolytic paratope extraction), or as an
intact molecule that will be digested by a protease after the immune complex is
formed (proteolytic paratope excision). The characterization of antibody-paratopes is
Complex
formation
Proteolytic
digestion
Intact
antigen Intact
antigen
Complex
formation
Proteolytic
digestion
Epitope excision Epitope extraction
Washing Dissociation
MS MS
Figure 5: The principle of mass spectrometric epitope identification. (a) For epitope excision the
antigen is bound to the immobilized antibody and digested with proteases. Unbound
peptides are washed off and the affinity bound peptides are dissociated from the antibody.
Both fractions are analysed by mass spectrometry. (b) In epitope extraction, the antigen is
first digested in solution, and the proteolytic fragments are allowed to bind to the antibody.
Introduction 10
considerably more difficult due to i) higher stability to proteolytic digestion of the
antibody compared to the antigen, which makes difficult the application of the
proteolytic excision of the paratope; ii) the fact that the antigen binding sites might be
scattered within the 6 complementarity determining regions (CDRs) of the heavy and
light chains and the individual sequences resulted by digestion in solution during
epitope extraction might not display affinity to the antigen and iii) the lack of genomic
data for most of the antibodies to provide fast identification of the paratope
sequences by comparing the set of peptide masses obtained in the mass spectrum
of the paratope elution with the theoretical masses from the sequence database. A
first example of paratope identification by mass spectrometry was described for the
camel anti-lysozyme antibody cAbLys3 (see Figure 6) [43]. Camel antibodies lack the
light chains, therefore containing only three CDRs [44]. The CDR3 is significantly
larger than those in human and mouse immunoglobulin and forms an exposed loop
of 24 amino acids that fits into the active site cleft of lysozyme [45]. Mass
spectrometric paratope identification was applied in this case for a 26 amino acid
synthetic peptide containing the CDR3 sequence.
Although mass spectrometry has been an established technique in organic chemistry,
the involatility of the macromolecules has limited the applications in the biological and
medical field in the past. This difficulty has been overcome by the introduction of “soft”
ionization techniques for effectively dispersing proteins and other molecules into the
gas phase with no or little fragmentation. The predominant methods are today i)
matrix-assisted laser desorption and ionization (MALDI), and ii) electrospray
ionization (ESI).
Lysozyme
cAbLys3
CDR3
Figure 6: Ribbon representation
of the non-covalent complex
formed by cAbLys3 camel
antibody and hen eggwhite
lysozyme (HEL). The CDR3
region of the cAbLys3 is marked
in yellow. The structure was
prepared with BAllView 1.1
based on the crystal structure
with the PDB accession number
1MEL.
Introduction 11
In MALDI mass spectrometry applications, the analyte is co-crystallized with a large
excess of a matrix. The matrix molecules are typically organic acids which have an
absorption in the wavelength at which the laser is used (UV, visible or infrared) [46,
47]. Pulses of laser light (1-10 ns) are applied to the surface of the sample, causing
desorption and ionization of the analyte-matrix mixture. The thermal-spike model
proposes that the energy absorbed by the matrix molecules causes rapid heating of
the irradiated layers of sample followed by evaporation of matrix together with the
analyte molecules. The ionization of the analyte occurs probably subsequent to the
ejection of the molecules from the support [48]. A schematic representation of the
MALDI process is illustrated in Figure 7a. Typically, the mass spectrum of a sample
ionized by MALDI contains singly charged molecular ions and ions of low charge
states.
In contrast to MALDI-MS, electrospray ionization (ESI) sources operate at
atmospheric pressure and provide the transfer of the ions present in liquid samples in
the gas phase as isolated entities [49]. Analyte ions are generated by solubilisation in
suitable solvents such as acidic aqueous solutions containing methanol or
acetonitrile. The analyte solution is pushed through a very small metal capillary. The
high electric field applied between the needle and the counter electrode forces the
solution to emerge from the tip of the needle giving rise to the so called Taylor cone.
If the electric field is high enough small charged droplets form. The formation of fine
droplets from the solution emerging from the needle is facilitated by a sheet flow of
nitrogen gas. According to the “ion-evaporation” model, the solvent from each droplet
evaporates yielding a higher charge density. When the Coulomb repulsion becomes
of the same order as the surface tension, the droplet undergoes fissions producing
smaller droplets that also evaporate. The process leads eventually to the formation of
droplets containing a single ion. Ultimately fully desolvated ions result from complete
evaporation of the solvent.
Introduction 12
a) b) Figure 7: (a) Schematic representation of the ion formation in MALDI mass spectrometry; (b)
Schematic representation of the electrospray process.
The advances in the development of ionization techniques led to an increasing
interest in the technological improvements of time-of-flight, ion trap and Fourier
transform mass spectrometers for applications in peptide and protein analysis. While
FTMS combines all the high performance characteristics (accuracy, resolution,
sensitivity), the high cost of magnets and maintenance and the complexity of
operation have limited their widespread use to industrial laboratories; however
several mass spectrometry research laboratories recently focused on extending the
applications and performance of the FTICR-MS methods. Triple quadrupole, TOF
and quadrupole ion trap mass spectrometers are three other types of mass analysers
with widespread use primarily owing to their cost and ease to use.
nebulising gas
Solution with
analyte
Atmospheric pressure
Taylor cone
Counter
electrode
+ -
Skimmer
Vacuum stages
To the mass
analyser
To the mass
analyser
A2
A3
A1
Analyt co-crystallized
with matrix on sample
target
Laser shot
Desorption plume
A1
A2
A3
H+
H+
H+
H+
H+
H+
H+
A2
A1
A3 H+
H+
H+
H+
H+
H+
H+
Matrix-analyt clusters
A2
A1
A3
H+
H+
H+
H+
H+
H+
H+
Analyt ions
Extraction grid
+ 20 kV
Introduction 13
1.4. Pathophysiological characteristics and therapeutic perspectives of
Alzheimer´s Disease
“She sits on the bed with a helpless expression. What is
your name? Auguste. Last name? Auguste. What is
your husband’s name? Auguste, I think.” [50]
It has been already a century ago since the German psychiatrist Alois Alzheimer
presented the case of Auguste D., a 51-year-old lady who had shown progressive
loss of cognitive functions and psychosocial competence. A. Alzheimer described for
the first time the clinical picture of presenile dementia as well as the histological
findings of amyloid plaques, neurofibrillary tangles and arteriosclerotic changes.
Alzheimer´s disease is clinically characterized by a progressive decline of cognitive
functions from mild forgetfulness and cognitive impairment, to widespread loss of
memory, language and logical thinking having impact on the ability to perform
everyday activities and changing the patient’s behavior. Death occurs, on average,
10 years after the diagnosis. In addition to its direct effects on patients, advanced AD
loads a tremendous burden on family caregivers and causes substantial nursing
costs for the society [51]. Due to the increase of life expectancy of the population, the
absolute number of people afflicted by AD is expected to grow substantially. It is
estimated that there are currently 26 million people worldwide suffering of
Alzheimer´s disease, and the global prevalence is expected to increase to more than
100 million by 2050.
Current medications approved for the treatment of Alzheimer´s disease are based on
the modulation of neurotransmission. Acethylcholinesterase (AchE) inhibitors attempt
to address the cholinergic deficits seen in AD and are used for mild to moderate
cases. Memantine an (N-methyl-D-aspartate)-receptor antagonist that has been used
for the treatment of moderate to severe Alzheimer dementia aims to prevent the
neuronal excitotoxic effect exerted by high levels of glutamate. Although producing
moderate symptomatic improvements of the cognitive function, none of these drugs
appears to be able to cure Alzheimer´s disease [52].
Introduction 14
Hence, an enormous need exists for the development of new medications for AD with
strong disease-modifying properties, and research is focused on the development of
new therapeutic strategies that target the underlying pathogenic mechanisms of
Alzheimer´s disease.
A comparative examination of the brains from AD patients and normal elderly
individuals reveals a dramatic loss of brain tissue [53]. Shrinkage of the brain is
extremely severe in the hippocampus, temporal and parietal lobes and is mainly
observed in the widened cortical sulci and ventricular dilatation as depicted in Figure
8b. The histopathological hallmarks of Alzheimer´s disease are loss of cholinergic
and glutamatergic neurons, intracellular and extracellular deposits of proteins and
microvascular angiopathy. Many neurons in the brain regions typically affected in AD
contain abnormal protein deposits called neurofibrillary tangles that occupy much of
the perinuclear cytoplasm (see Figure 8a). The neurofibrillary tangles consist of
microtubule-associated protein Tau in abnormally phosphorylated form [54]. The in
vitro phosphorylation of tau has been reported to inhibit the polymerization of tubulin
[55] into the microtubules. Microtubules are crucially important structures which run
through the cell and are involved in axonal transport, synaptic transmission, cell
support and shape.
a) b)
Normal Alzheimer´s Disease Normal Alzheimer´s Disease
Figure 8: Pathophysiological characteristics of Alzheimer´s Disease compared with a healthy
individual: a) neurofibrillary tangles and amyloid plaques; b) brain cross section
showing atrophy of the brain tissue affecting predominantly the language and
memory lobes. Copyright © 2000-2009 American Health Foundation. All rights
reserved.
Introduction 15
The extracellular deposits are referred to as neuritic or senile plaques and consist of
aggregated amyloid-ß protein [56] surrounded by astrocytes and neurites emanating
from local neurons. Microvascular angiopathy caused by the deposition of amyloid-ß
protein on the walls of the arterioles and venules was found outside the brain as well
as within the cerebral cortex of the brains from patients with Alzheimer´s disease [57,
58]. Cerebral amyloid angiopathy can lead to hemorrhages which may contribute to
the cognitive decline [59]. The hypothesis that states the fundamental role of the
overproduction and accumulation of Aß in senile plaques in the pathology of AD has
been extesively studied in the last two decades. An overview concerning the origin of
amyloid-ß protein and the accumulation in senile plaques as well as the main
therapeutic strategies that are currently pursued will be discussed in the following
sections of the introduction. In Alzheimer´s disease, excessive activation of NMDA
receptors by L-Glutamate (L-Glu) is believed to cause elevated cytosolic Ca2+ which
then initiates pathological events that ultimately lead to neurodegeneration [52, 60].
Amyloid-ß (Aß) was first sequenced from the meningeal blood vessels of AD patients
and individuals with Down syndrome by George G. Glenner [58, 61] and then
identified in the senile plaques [62]. Aß is proteolytically cleaved from the amyloid
precursor protein (APP) that contains a single transmembrane domain, with a longer
N-terminal amino acid sequence emanating out of the cell and a shorter C-terminal
domain jutting into the cytosol. APP is encoded by a gene located on the
chromosome 21 [63-65] and although is produced by many cells and tissues its
precise biological role has remained unknown. Several forms of APP that differ
mainly at the amino-terminal end of the sequence have been described to arise by
alternative splicing: APP-695, APP-751 and APP-770 [66-68]. The enzymes that play
a central role in the proteolytic processing of APP are α-, β- and γ-secretases (see
Figure 9). The proteolytic cleavage by α-secretase occurs 12 amino acids NH2terminal to the transmembrane domain and releases a large soluble fragment (αAPPs) into the extracellular space. The 83-amino acid residue COOH-terminal
fragment is retained in the membrane and is further cleaved by γ-secretase,
generating the p3 peptide fragment and a 57/59 amino acid residue carboxy-terminal
fragment (CT57/59). Alternatively, APP is cleaved 16 amino acids N-terminal to the
α-secretase cleavage site by ß-secretase releasing ß-APPs into the extracellular
space and retaining a 99-amino-acid residue in the membrane. The cleavage by γIntroduction 16
secretase produces a 40/42 peptide fragment referred to as Aß and the CT57/59.
The α-secretase activity was described to be exerted by three related
metalloproteases of the ADAM (a disintegrin and metalloprotease) family, ADAM-9
[69], ADAM-10 [70] and ADAM-17 [71]. Two aspartyl enzymes responsible for the ßsecretase cleavage have been identified in 1999 referred to as BACE (ßAPP
cleaving enzyme) [72-75] and BACE-2 [76, 77]. BACE activity can also generate
fragments of APP cleaved at secondary sites such as Glu11 within the Aß sequence
[78]. The second enzymatic activity required for Aß generation is exerted
heterogeneously by γ-secretase. Most of the full-length Aß species produced is a 40residue peptide (Aß40), whereas a smaller proportion is a 42-residue carboxyterminal form.
Figure 9: Proteolytic processing of APP. The conjoint cleavage of APP by α- and γ-secretase
produces the harmless fragment p3, the carboxy-terminal C57/59 and the longer soluble
α-APP; Alternatively the cleavage by ß- and γ-secretase releases the 40/42 residues long
fragment called Aß that is prone to aggregation.
Under normal circumstances, Aß generated in the CNS is cleared with a half-life of 12 h [79]. Initially, Aß42 which is more prone to aggregation than Aß40 is deposited in
diffuse (nonfibrillar) plaques with little or no detectable neuritic dystrophy. The
mechanism through which the aggregated Aß exerts its toxic effects is still
controversial. It was shown that aggregated forms of synthetic Aß peptides can
H2N COOH APP770
Kunitz protease
inhibitor domain
Ox-2 antigen
domain
289 345 364 672 713
αβ γ
sAPPα C83
p3 sAPPβ C99
α-secretase
672DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA713
β α γ γ
γ-secretase γ-secretase
β-secretase
Introduction 17
cause damage to cultured neuronal cells [80, 81]. However, more recent findings
suggest that soluble oligomeric prefibrillar forms of Aß may represent the neurotoxic
species that causes neurotoxicity and synaptic dysfunction [82, 83].
The dominating hypothesis concerning the mechanisms leading to Alzheimer’s
disease assigns a central role of the accumulation of Aß in brain to the initiation of a
cascade of pathological events that ultimately lead to neurodegeneration and
dementia [52, 84]. A first argument supporting a causal role of Aß in Alzheimer´s
disease came from the identification of the APP gene locus on chromosome 21 and
the earlier finding that individuals affected by Down syndrome posses 3 copies of the
chromosome 21 and develop invariably AD pathology. Additionally, several mutations
that are responsible for early-onset forms of familial Alzheimer’s disease (FAD) have
been identified in the APP gene. These mutations are located directly adjacent close
to the ß- and γ-secretase cleavage sites favoring the proteolytic processing of APP
and leading to increased Aß production. Mutations in the genes encoding PS1 and
PS2 are also responsible for elevated production of Aß. Increased Aß plaque
deposition in brain has been associated with the presence of apolipoprotein E
(apoE)-protein although the mechanism remains unknown [85]. Recent data showed
a reduction of Aß deposition in the offspring by crossing APP overexpressing
transgenic mice with apoE-deficient mice [86].
1.5. Immunotherapeutic strategies for Alzheimer´s Disease
A primary goal of research on Alzheimer´s disease is to develop therapeutics able to
prevent or reverse the cognitive decline of AD patients. As discussed in the previous
section the most studied hypothesis emphasizes the neurodegenerative effect
exerted by amyloid deposits. Based on the current knowledge provided by these
studies, the development of anti-Aß therapeutics appears as a rational approach for
treatment.
Present approaches are focused on (i) the modulation of Aß production, (ii)
preventing Aß aggregation and (iii) clearance of soluble Aß and amyloid deposits
from brain (see Figure 10). The attempt to reduce the production of Aß led to the
Introduction 18
development of potent inhibitors that block the activity of ß- and γ-secretase.
Although the inhibiting compounds have been tested in mice and were effective in
reducing Aß, testing in humans has been barely attempted due to the concern
regarding potential side effects. Preventing the formation of Aß aggregates has been
attempted by chelating the metal ions (Zn2+, Cu2+) reported to enhance Aß deposition
[87-89].
The idea of using the ability of the immune system to produce specific antibodies that
recognise soluble Aß or amyloid deposits and lead to their clearance from brain has
gained increasing interest in recent years. Several immunotherapeutic strategies
have been under investigation including active immunization with synthetic Aß,
protofibrillar Aß assemblies or Aß peptide fragments conjugated to a carrier protein,
and passive immunization with monoclonal Aß-specific antibodies [90, 91]. The
studies carried out by B. Solomon and collaborators provided first evidence that
antibodies recognizing Aß were effective in blocking the formation of amyloid fibrils in
vitro [92], dissolving pre-existing amyloid fibrils [93] and preventing neurotoxicity of
Aß fibrils. These observations performed in vitro were followed by the immunization
of transgenic mice overexpressing APP with Aß. The first results of immunization
with Aß42 showed a dramatic reduction of fibrillar Aß deposition in young PDAPP
APP

ß
γ


Amyloid plaque
Production
Clearance
Aggregation
Clearance
ß, γ-secretase
inhibitors
Aggregation
inhibitors
ImmunotherapyImmunotherapy
Reduced levels of
Aß in brain
Figure 10: Schematic presentation of the therapeutic strategies aiming to prevent and reverse the
pathological events leading to amyloid plaque formation.
Introduction 19
transgenic mice, and reduction or even reversal of amyloid deposition if the
immunization was performed in older animals [94]. A correlation between plaque
reduction and the ability to perform memory tasks was not possible due to the
observation that in PDAPP mice cognitive impairment precedes amyloid deposition
[95]. However, improvements in cognitive function associated with plaque reduction
were described in two parallel studies in which TgCRND8 transgenic mice were
immunized with protofibrillar Aß42 [95] and APP/PS1 mice with Aß42 [96]. Both
studies did not observe any adverse effect of the vaccine. On the basis of the
promising preclinical findings, a first clinical trial was initiated in which 300 patients
received active immunisation with AN1792 (Aß42 and adjuvants). Unfortunately, 6%
of the patients developed symptoms of meningoencephalitis and the trial was
suspended [97]. At present, the inflammatory response encountered is attributed to
the infiltration of the brain with activated T cells [98, 99]. However, the patients with
high anti-Aß titers had significantly less deterioration of cognitive performance in the
year following the clinical trial, than patients with little or no anti-Aß antibodies [100,
101].
In recent years, the immunotherapeutic studies have been concentrated on the
development of passive immunization strategies. The intravenous administration of
amyloid specific antibodies might offer potential advantages over the active
immunization approach: (i) avoid the variability of the immune response across the
individuals receiving the immunogen by providing known amounts of antibody of
known epitope specificity; (ii) do not trigger T cell activation; and (iii) can be
withdrawn if adverse reaction are encountered [102]. A number of controversies have
arisen regarding the antibody specificity for passive immunization studies. While Bard
et al. showed that only N-terminal domain antibodies are able to clear amyloid
plaques [103], DeMattos et al. argue that an antibody to the Aß mid-domain with little
reactivity to brain amyloid might be effective [104]. Alternatively, Morgan et al.
reported that passive administration of antibodies specific to a carboxy terminal
domain of Aß was able to reverse cognitive deficits in transgenic mice.
Several mechanisms have been proposed to explain the therapeutic action of antiamyloid antibodies (see Figure 11). The plaque breakdown hypothesis relies on the
ability of a small amount of Aß-antibodies (0.1%) to cross the blood-brain barrier into
Introduction 20
the CNS, bind the neuritic plaques and promote Fc-receptor-mediated phagocytosis
of the plaques (Figure 11a). According to the results obtained by active immunization,
antibodies able to recognize the Aß-plaques target the N-terminal domain of Aß. The
epitope recognized by such “plaque-specific” antibodies was elucidated by our
laboratory to comprise the Aß(4-10) sequence [16]. The peripheral sink hypothesis is
supported by the experiments of DeMattos and coworkers and Morgan and
colleagues. Peripheral administration of antibodies that bind monomeric Aß causes
the transport of Aß from the CNS to plasma until the equilibrium is reached (see
Figure 11b). A third possible mechanism for the action of anti-Aß antibodies suggests
a catalytic transformation of Aß peptide into a structure less compatible with amyloid
fibril formation (Figure 11c).
a) Plaque breakdown b) Peripheral sink c) Aggregation inhibitor
Brain
Plasma
Neuritic
plaque
Monomeric Aß
Microglia
Anti-Aß
antibodies
Figure 11: Schematic representation of proposed immunotherapeutic actions of anti-Aß antibodies (a)
entry of anti-Aß antibodies to the brain could result in decoration of amyloid plaques by
antibodies and subsequent removal by microglia activated through Fc receptor
mechanisms; (b) antibodies to Aß act as a “sink” for Aß as it moves from the CNS to
plasma; (c) antibodies to Aß may act to block interactions between monomeric Aß
preventing aggregation.
Introduction 21
A molecular understanding of the neuroprotective effect of the Aß-specific antibodies
can be expected by the elucidation of the antigenic determinant targeted by the
active antibodies as well as by the structural characterization of these antibodies. A
fast and reliable tool for epitope identification is provided by the mass spectrometric
methods in conjunction with affinity chromatography and proteolytic assays.
1.6. Scientific goals of the dissertation
Recent advances in immunology and molecular biology have lead to the
development of therapeutic vaccines which are of potential use in chronic diseases
such as cancer, cardiovascular disorders and neurodegenerative diseases, where
efficacies of available therapies are poor. Clearly, future developments in vaccine
design will rely substantially on a more complete understanding of the structural
basis of immune response. The application of mass spectrometry as a molecular tool
in biological and medical sciences has rapidly increased with the development of soft
ionization techniques that enable ionization of macromolecules without structure
alterations. Due to the low sample amounts required for analysis, relatively high
speed and molecular specificity of measurement, mass spectrometry can be used in
combination with analytical-scale separation methods of the protein of interest.
The major scientific objectives of the dissertation comprising several parts have been
summarized as follows:
1. Molecular characterisation of Aß plaque specific antibodies. In this part (i) the
examination of the contribution of each residue of the plaque specific Aßepitope to the interaction with the cognate antibodies, (ii) the effect of zinc ions
on the binding of amyloid peptides were investigated by ELISA.
2. Epitope elucidation of naturally occurring Aß-reactive antibodies from human
serum. The major goals of this study were (i) the isolation Aß-specific
antibodies from human serum immunoglobulin preparations; (ii) the epitope
elucidation of the Aß-autoantibodies by proteolytic epitope excision combined
with mass spectrometry. Furthermore, the reactivity of ß-amyloid peptides to
Introduction 22
the antibodies was examined and the clonal heterogeneity of the human
serum Aß antibodies was investigated by mass spectrometric proteome
analysis.
3. Investigation of the cleavage specificity of HtrA1 protease using ß-amyloid
peptide substrates.
4. Mass spectrometric and immunoanalytical epitope identification of an antibody
against the H1-carbohydrate recognition domain of the asialoglycoprotein
receptor. The major goals of this part were (i) the analysis of the primary
structure of H1 using proteolytic digestion in conjunction with mass
spectrometry, (ii) the identification of the epitope using proteolytic excision of
the antigen and mass spectrometry.
Results and discussion 23
2. RESULTS AND DISCUSSION
2.1. Characterisation of Aß-plaque specific antibodies
2.1.1. Metodology of mass spectrometric epitope identification
To identify an antigenic determinant recognized by an antibody, both epitope
excision and extraction have been employed. In the first step of the epitope
excision approach, a solution containing the intact antigen is exposed to the
immobilized antibody. The immune complex is allowed to form and the proteins
remaining in solution are removed and collected as non-binding fraction (Figure
12a). The affinity matrix is washed to ensure complete removal of free antigen. In
the second step the specific proteolytic enzyme is applied and allowed to cleave
the amino acid residues that are not shielded by the interaction to the antibody.
The proteolytic fragments released in the supernatant fraction are collected to be
analyzed by mass spectrometry.
In the third step the matrix is washed extensively in order to ensure complete
removal of contaminant peptides. The last volume of the washing buffer referred to
as washing fraction is collected to be analyzed by MS. The amount of buffer
appropriate for a complete removal of contaminant fragments is determined in a
preliminary experiment. The peptides remaining bound to the antibody are eluted
under acidic conditions (elution fraction). Due to the use of solutions with high salt
concentration for the washing, the pH of the elution fraction is verified and the step
repeated in case of higher pH. To further use the column, the affinity matrix is
regenerated by washing with the elution solution and with neutral buffer.
In epitope extraction the antigen is proteolytically digested in solution and the
peptide fragments resulting are applied to the column and allowed to react with the
antibody. The supernatant fraction containing non-bound peptides is collected.
Usually the supernatant of the epitope extraction contains non-epitope fragments
but also epitope fragments if present in excess compared to the binding capacity
of the antibody. Prior to elution, the column is washed and the washing fraction is
collected in order to test whether the column is free from contaminant peptides.
Results and discussion 24
a) b) Figure 12: Schematic representation of the methodology for epitope excision a) and epitope
extraction b) using immobilized antibody
The peptides that are affinity-bound to the antibody are dissociated and the affinity
media regenerated for further use. For mass spectrometric analysis of the epitope,
the affinity-bound peptides can be eluted from the antibody using 0.1%
trifluoroacetic acid [16, 42], 4M MgCl2 [33], 0.1M glycine (pH 2.3) [35, 105]. The
0.1% trifluoroacetic acid was the solvent of choice in this work due to the
compatibility with the mass spectrometric methods of peptide identification.
Alternatively, the peptides remaining bound to the immobilized antibody after
epitope excision or extraction can be analyzed by applying an aliquot of the affinity
media containing the immune complex directly onto the target. The antigen is
dissociated from the antibody on the target by the addition of the acidic MALDI
Intact
antigen
Complex
formation
Removal of nonepitope fragments
Epitope elution
Affinity media
regeneration
Washing
Supernatant Elution fraction
Proteolytic
digestion
Proteolytic
fragments
Removal of nonbound antigen
Complex
formation
Removal of nonepitope fragments
Proteolytic
digestion
Epitope elution
Affinity media
regeneration
Washing
Intact
antigen
Non-binding fraction Supernatant Elution fraction
Results and discussion 25
matrix [35, 106, 107] prior to the mass spectrometric analysis. This procedure
allows the use of the remaining affinity media containing the antibody-antigen
complex for proteolytic experiments in order to reduce the peptides bound to the
antibody to the minimal sequence maintaining affinity.
The choice of the protease depends on the sequence of the antigen and the length
of the cleavage products. By overlapping the fragments identified in the elution
fraction upon treatment with various proteases, a precise identification of the
epitope amino acid sequence is achieved.
The epitope excision and extraction method can take place in solution [34], or by
using an immobilized antibody. If the experiment is carried out in solution, the
methodology implies the use of centrifugal devices provided with appropriate
membranes to separate the immune complex from the unbound peptide fragments.
The covalent immobilization of the antibodies on insoluble matrices enables the
extensive wash of the affinity media for unspecific bound peptides removal. The
matrix should be chemically and physically inert and provided with a spacer arm in
order to overcome any steric hindrance between ligand and target molecule.
Ligands are coupled via reactive functional groups to Sepharose beads [16, 33, 42,
106] or magnetic nitrocellulose beads [105]. Affinity capture of the specific
antibody using Sepharose beads coupled to an Fc-specific antibody has been also
reported [108].
2.1.2. Mass spectrometric elucidation of the epitope recognized by
polyclonal anti-Aß42 and monoclonal anti-Aß(1-17) antibodies
Immunisation of transgenic mouse models of Alzheimer’s disease (AD) with ßamyloid peptide (Aß) of 40 to 42 amino acids led to the production of antibodies
that were found to inhibit and disaggregate Aß-fibrils, and were able to reduce ADrelated neuropathology and memory impairments [16]. The mechanism underlying
these therapeutic effects remained unclear although the elucidation of the epitope
recognised by these antibodies provided important information.
Results and discussion 26
Using epitope excision in combination with mass spectrometry, the epitope
recognised by the therapeutically active antibodies was identified as the N-terminal
Aß(4-10) sequence [16, 90]. A series of proteolytic enzymes was employed
(trypsin; α-chymotrypsin; Glu-C) all of which provided the corresponding Nterminal Aß-epitope fragments. Trypsin provided cleavage after Lys-16 but not
after Arg-5 indicating that Arg-5 is part of the epitope (see Figure 13); GluC was
able to cleave the Aß(1-40) in complex with the antibody at Glu-3 and Glu-11
indicating the sequence [3-10] as epitope. Additional information was provided by
α-chymotrypsin which cleaved after Tyr-10 but not after Phe-4 suggesting that
Phe-4 is part of the epitope. The results of the epitope identification using these
proteases ascertained unambiguously the minimal epitope, Aß(4-10) (FRHDSGY).
Epitope excision-MS studies with the polyclonal Aß-antibodies were performed
with synthetic Aß(1-42), Aß-aggregates, brain tissue and mouse plaques, all of
which provided the identical Aß-N-terminal epitope; in addition the specificity and
affinity of the plaque-specific epitope were ascertained by ELISA and alanine
mutation studies with the synthetic Aß-epitope peptides [109].
Epitope identification was also carried out with a commercially available
monoclonal antibody anti-Aß(1-17) using proteinase K. In Figure 14 the arrows
indicate the cleavage sites observed by proteolytic digestion of Aß(1-40) with
proteinase K in solution. Considering the sequence [1-17], used as immunogen to
produce the antibody, the enzyme was able to cleave in solution after Ala-3, Phe-4,
His-5, Tyr-10, His-13. The elution fraction collected after the proteolytic digestion
Figure 13: Schematic representation of the results obtained by the mass spectrometric
identification of the epitope recognized by the polyclonal anti-Aß42 antibody. The
sites of proteolytic cleavage determined in the eluted fragments from epitope
excision experiments with trypsin, GluC and α-chymotrypsin are marked with filled
arrow. The cleavage sites that were not accessible to the proteolytic cleavage by
each of the enzymes used are marked with dashed arrow.
DAEFRHDSGYEVHHQKLVFFAEDVGSNHGAIIGLMVGGVVIA
4FRHDSGY10
Trypsin
GluC
α-chymotrypsin
Results and discussion 27
of the immune complex contained the sequence [3-10]. This result shows that
both polyclonal anti-Aß(1-42) and the monoclonal anti-Aß(1-17) antibodies target
an identical epitope sequence.
As described in the Introduction, the immunotherapeutical approach in Alzheimer´s
Disease is based on the antibody´s ability to bind amyloid beta peptides
preventing or blocking the formation of the amyloid plaques. The antibodies that
target the N-terminal domain of Aß are able to recognize specifically not only the
soluble Aß but also the Aß molecules within amyloid plaques. In the amyloid fibrils,
the N-terminal domain of Aß is not involved in intermolecular interactions that
stabilize the fibril, being exposed at the surface of the fibrils. A structural model of
the amyloid fibrils will be discussed in par. 2.1.4. The polyclonal anti-Aß(1-42)
antibodies that target the Aß(4-10) epitope were shown to inhibit Aß fibrillogenesis
as well as the cytotoxic effects of Aß related to the formation of small oligomers
and protofibrils. Moreover these antibodies were able to disrupt preformed Aß(142) fibers and to recognize Aß in amyloid plaques [16]. These results indicate that
antibodies to the N-terminal sequence might have therapeutic applications in
humans. Figure 15 shows schematically the proposed mechanism of Aß plaque
clearance based on the immunization with Aß(1-42).
Figure 14: Schematic representation of the results obtained by the mass spectrometric identification
of the epitope recognized by the monoclonal anti-Aß(1-17) antibody. The arrows depict
the sites of proteolytic cleavage determined in the supernatant of the epitope excision
with Proteinase K. The elution fraction contained only the fragment [3-10].
DAEFRHDSGYEVHHQKLVFFAEDVGSNHGAIIGLMVGGVVIA
3EFRHDSGY10
Proteinase K
Results and discussion 28
2.1.3. Identification of functional amino acid residues within the ß-amyloid
plaque specific epitope using alanine-scanning mutagenesis
The mass spectrometric approach to the elucidation of the epitope recognized by
the polyclonal anti-Aß(1-42) antibodies lead to the identification of the N-terminal
sequence Aß(4-10) (FRHDSGY). The same antigenic determinant was identified
by mass spectrometric epitope indentification in the case of the monoclonal
antibody anti-Aß(1-17). In order to identify the relative contributions of each of the
individual amino acids comprised by the epitope to the interaction with the
antibodies, the reactivities of the antibodies towards synthetic peptides containing
alanine single-site mutations were investigated by ELISA.
ß-secretase
Extracellular domain Transmembrane
domain
EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKK
γ−secretaseα-secretase
APP
1 42
ß-secretase and
γ-secretase cleavage
aggregation
Anti-(N-terminal) IgG
Aß(1-42)
1 40
1 40
140
1 40
140
1
40
1
40
Figure 15: Proposed mechanism of action of the plaque-specific antibodies. N-terminal
specific antibodies are able to bind amyloid fibrils due to exposed N-terminal
domain of Aß. Plaque breakdown by microglia activated through Fc receptor
mechanisms could explain the Aß plaque clearance effect of these antibodies.
Results and discussion 29
Biotinylated Aß(1-10) peptides, both wild type and mutant constructs with a Nterminal pentaglycine spacer, were prepared by Solid Phase Peptide Synthesis
(SPPS) according to the Fmoc strategy. The synthesis protocol was as follows: (i)
DMF washing (3 x 1 min), (ii) deprotection with 20 % piperidine in DMF (15 min),
(iii) DMF washing (6 x 1 min), (iv) coupling of 5 equivalents of Fmoc amino acid
using PyBOP and NMM in DMF (50 min), (v) DMF washing (3 x 1 min). After
completion of the syntheses and removal of the N-terminal Fmoc protecting group,
the free amino group was biotinylated using 5 equivalents of D-(+)-biotin. Then the
peptides were cleaved from the resin using a cleavage solution containing 95 %
TFA as cleavage reagent and 2.5 % triethylsilan as scavengers. After cleavage,
the solution containing the resin and free peptide was filtrated to remove the resin
and washed twice with TFA. The peptide present in the filtrate was precipitated
using 10 volumes of cold tert-butyl-methyl-ether over the volume of filtrate. The
precipitate was filtrated, then the solid material was washed three times with
diethylether (10 ml) and dissolved in 5 % acetic acid (aqueous solution) prior to
freeze-drying. The crude products were purified by RP-HPLC and analysed by
mass spectrometry.
Table1 shows the characteristics of the synthetic peptides used in the alanine
scanning mutagenesis experiment. To allow a comparable extent of immobilization
to the microtiter plates the peptides were synthesized with a biotin residue at the
amino-terminal end. The binding of the peptides to the plates is mediated by the
interaction of the biotin with the streptavidin coated to the plates. A pentaglycine
spacer was placed between biotin and the Aß(1-10) sequence to ensure the
accessibility of the epitope to the antibody. The molecular masses of the
synthesized peptides were determined using an ESI-FTICR-MS. The measured
masses exactly matched the predicted molecular weights indicating that the
correct sequences were obtained in the synthesis of the peptides. The purity of the
peptides was assessed by analytical RP-HPLC and mass spectrometry. For
analytical RP-HPLC an analytical Nucleosil 300-7 C18 column (Macherey-Nagel,
Düren, Germany) was used as stationary phase.
Results and discussion 30
Table 1: Characteristics of mutant and wild type Aß-epitope peptides. Linear gradient elution (0min
0 %B; 5 min 0 % B; 50 min 90 % B) with eluent A (0.1 % TFA in water) and eluent B (0.1 %
TFA in (80 % acetonitrile, 0.1 % TFA in water) was employed at a flow rate of 1 mL/min at
ambient temperature. Peaks were detected at λ=220 nm. The samples were dissolved in
eluent A.
Peptide
No.
Mutation Sequence
HPLC
Rt (min)
[M+H]+
calculated found
1 WT Biotin-GGGGGDAEFRHDSGY 23.63 1706.6976 1706.7367
2 F4A Biotin-GGGGGDAEARHDSGY 21.15 1630.6663 1630.6948
3 R5A Biotin-GGGGGDAEFAHDSGY 24.14 1621.6336 1621.6540
4 H6A Biotin-GGGGGDAEFRADSGY 24.04 1640.6758 1640.6977
5 D7A Biotin-GGGGGDAEFRHASGY 24.40 1662.7078 1662.7124
6 S8A Biotin-GGGGGDAEFRHDAGY 23.79 1690.7027 1690.7340
7 G9A Biotin-GGGGGDAEFRHDSAY 23.97 1720.7132 1720.7395
8 Y10A Biotin-GGGGGDAEFRHDSGA 22.69 1614.6714 1614.7321
9 (F4-Y10)A Biotin-GGGGGDAEAAAAAAA 20.8/23.54 1341.5852 1341.5614
All mutant peptides were immobilized on microtiter plates at a fixed concentration
(1 ng/µl). After blocking, the antibodies were added in 8 serial twofold dilutions
using stock solutions of 1 µg/µl. The bound anti-amyloid antibody was detected by
an alkaline phosphatase-conjugated antibody as described at the Experimental
Section. In order to provide accurate background substraction, triplicate wells of
each antibody dilution without antigen were used as shown in Figure 16.
As expected, the response curves were significantly different for the polyclonal
and the monoclonal antibodies although the antibodies target the same epitope
Aß(4-10). The wild type construct (depicted with filled squares) reacted in a dosedependent manner with both antibodies Figure 16 a) and b).
Results and discussion 31
a) b) Figure 16: Dose-response curves of the alanine scanning mutagenesis experiment: a) reactivity of
wild type Biotin-(G)5-Aß(1-10) (filled square) and of the alanine mutated constructs
towards polyclonal anti-Aß42 antibody; b) reactivity of wild type Biotin-(G)5-Aß(1-10)
and of the alanine mutated constructs towards monoclonal anti-Aß(1-17) antibody. The
insert in the figure 16b shows a detailed view of the response shown by the alanine
mutated constructs. Background signals from wells without antigen were substracted.
0
200000
400000
600000
800000
1000000
1200000
1000200040008000160003200064000
0
5000
10000
15000
20000
25000
30000
1000200040008000160003200064000
Antibody dilution
Fl uo re sc en ce (
45
0/
53
5
nm )
Fl uo re sc en ce (
45
0/
53
5
nm )
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
25050010002000400080001600032000
Antibody dilution
Fl uo re sc en ce (
45
0/
53
5
nm )
WT F4A
R5A
H6A
D7AG9A Y10A
S8A
Fl uo re sc en ce (
45
0/
53
5
nm )
Results and discussion 32
In the case of the polyclonal anti-Aß(1-42) antibody the ELISA fluorescence values
of all the mutant peptides obtained for the antibody dilution 1:250 were normalized
to the wild-type construct. Therefore, the binding ability of the mutant peptides is
depicted in Figure 17 as percentage of the binding of Biotin-(G)5-Aß(1-10).
According to the experimental results the alanine mutants can be sorted into 4
groups. A first group contains the mutants D7A and S8A which display binding
properties to the polyclonal anti-Aß(1-42) antibody close to that of the wild type
peptide. The Y10A can be considered as a particular example because it
possesses a 2.8 times lower binding ability of that of the wild type without
abolishing the binding. Mutations F4A, R5A and H6A led to complete abrogation of
antibody binding. In contrast, the replacement of glycine with alanine increased
slightly the binding ability of the mutant peptide.
As depicted in Figure 18 the results show a substantial difference between the
functional amino acid residues recognized by the antibodies. In contrast to the
polyclonal antibody, the reactivity of the epitope [4-10] towards the monoclonal
antibody was abolished by all single site alanine mutations introduced in the
WT F4A R5A H6A D7A S8A G9A Y10A
0%
20%
40%
60%
80%
100%
120%
Biotinylated peptides
bi nd in g ab ili
ty (%
o f W
T
)
F HR D S G YBiotin-(G)5- - COOH
bi nd in g ab ili
ty (%
o f W
T
)
Figure 17: ELISA-detected reactivity of alanine mutants to the polyclonal anti-Aß(1-42) antibody.
The relative immunoreactivities of the wild type and mutant peptides to the polyclonal
antibody were assessed by an indirect solid-phase ELISA as described in the
Experimental Section. The panel depicts the binding capability (in percent of that of
the wild type peptide) for all the mutants at an antibody concentration of 1 µg/ml.
Error bars represent the S.D. of triplicate determinations.
Results and discussion 33
sequence. Structural data were from [110] and the ribbon representation of the
structure was prepared with BALLWiew v1.1.1. .
The ELISA was effective to discriminate among the mutants of the Aß(4-10)
epitope showing that the residues Phe4, His5 and Arg6 are crucial for the binding
to the polyclonal anti-Aß(1-42) antibody, while the other four residues are less
important. This antibody was obtained by active immunization of transgenic AD
mice with Aß(1-42) oligomers and was shown to disaggregate preformed Aß-fibrils.
Interestingly, the 3-6 amino acid sequence located in the N-terminal region of Aß
(Glu-Phe-Arg-His) has been found previously as the epitope of 2 different antiaggregating antibodies [111].
F
R
H D
S G
Y
MonoclonalPolyclonal
F
R
H D
S G
Y
Phe4
His5
Arg6 Phe4
His5
Arg6
Asp7
Tyr10
Gly9
Ser8
Figure 18: Location of residues affecting the epitope binding specificity to the antibodies.
Schematic comparison of the critical amino acid residues (dark grey) within the
Aß(4-10) epitope involved in the interaction with a) the polyclonal anti-Aß(1-42) and
b) the monoclonal anti-Aß(1-17).
Results and discussion 34
2.1.4. Effect of zinc ions on the recognition of N-terminal ß-amyloid epitope
by plaque specific antibodies
Zinc is an important catalytic and structural component of many proteins in human
brain as well as a modulating agent in synaptic transmission, but is neurotoxic at
high concentrations [112]. High levels of zinc ions have been detected within
amyloid deposits [113, 114], which could result (i) from zinc-induced amyloid
deposition [89, 110, 115], (ii) from its accumulation as a protective agent after
amyloid deposition, or (iii) from a disruption of zinc homeostasis associated with
AD. In support of the Zn2+ mediated stabilization of Aß amyloid fibrils, solubilisation
of Aß from Alzheimer´s disease brain tissue has been found to be significantly
enhanced by the presence of metal chelators such as EGTA, N,N,N´,N´-tetrakis(2pyridyl-methyl) ethylene diamine and bathocuproine [116]. In recent studies the
metal chelator clioquinol has been reported to inhibit the aggregation of Aß [87]
and currently there are studies that investigate the effects of clioquinol
administered to transgenic mice developing amyloid plaques.
Different structural models propose that the Aß conformation within amyloid fibrils
contains two parallel ß-strands formed by the residues 18-26 (ß1) and 31-42 (ß2),
separated by a 180° bend formed by residues 27-30 [117]. The ß-strands form 2
layers of intermolecular, parallel ß-sheets. The BallView 1.1.1 program was used
to depict the three-dimensional structure of the Aß fibril based on the structural
data available at the Protein Data Bank with the accession number 2beg (Figure
19). By contrast, considerable NMR data obtained on Aß argue that the soluble
monomeric peptide has an unordered conformation in aqueous solution and
mainly adopts an α-helical structure in membrane-mimicking media [118]. The
residues 1-17 that form the N-terminal region of Aß are disordered and are omitted
from the structural model of the amyloid fibril. These residues constitute the outer
wall of the fibril and are not involved in the interactions of Aß within the fibrils, thus
remaining accessible to the interaction with partners critical for the pathology or
with therapeutic agents. The structure model of the N-terminal region in the
presence and absence of zinc ions will be discussed at the end of this section.
Results and discussion 35
Figure 19: Structure model of amyloid fibril indicating the core structure of residues 17-42. The
direction of the fibril axis is indicated by an arrow. In the ribbon diagram of the Aß(1742) the ß-strands are indicated by blue arrows and the nonregular secondary structure
is depicted by green spline curves. The hydrophobic, polar, negatively charged,
positively charged amino acid side chains are shown in grey, green, red and blue. The
ß-strands are indicated by blue arrows and the loop connecting the 2 ß-strands is
depicted by green spline curve. A schematic view of the fibril is shown in the insert on
the upper right side.
Based on the mass spectrometric fragmentation patterns of Aß(1-16)-Zn2+
complexes in the gas phase and the NMR data of the complexes in aqueous
solution at pH 6.5, the Aß(1-16) sequence has been identified as the minimal zincbinding domain. The residues His-6, His-13, His-14 and the Glu-11 carboxylate
were identified as ligands that tetrahedrally coordinate the zinc ions [109, 110,
119-122].
As described in the Introduction, the N-terminal region of Aß represents an
attractive therapeutic target for active and passive immunization approaches. Only
the antibodies raised against the N-terminal part of Aß are able to reduce the
plaque burden and restore cognitive deficits in mice models of AD. With the
assumption that active anti-Aß-antibodies and zinc ions target a similar or identical
domain of Aβ [16], the Aβ-epitope recognition by two different antibodies in the
presence of zinc was investigated in the present work. The mAb anti(1-17) clone
L17
V18
F19
F20
A21
E22
D23
V24
G25
S26N27
K28
G29
A30
I31
I32
L34
M35
V36
G37
G38
V39
V40
I41
G33
Fibril axis
A42
1DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA42
142
Fibril axis
Results and discussion 36
6E10 was chosen due to the previous identification of the Aß(4-10) epitope
specificity by a combination of affinity chromatography, proteolytic digestion and
mass spectrometric experiments performed in our laboratory. As a second
antibody, mAb-anti(1-40) clone Bam-10 targeting an epitope residing within the
amino acids Aß(1-12) was studied.
To investigate the effects of zinc ions on the binding of the antibodies to the Nterminal domain of Aß, a sandwich ELISA was designed. The Aß(1-16) peptide
was synthesized with a pentaglycine spacer and a biotin attached at the aminoterminal end. As a negative control for the binding of the zinc ions, biotin-(G)5Aß(1-10) was employed. This peptide does not contain the minimal zinc binding
domain but comprises the N-terminal epitope of the antibodies as previously
described (s. par. 2.1.3.). An example of the HPLC profile and MALDI-FT-ICR
mass spectrum of the pure Biotin-(G)5-Aß(1-16) peptide is depicted in Figure 20.
a) b)
A detailed description of the experimental conditions and a schematic
representation of the sandwich ELISA are given in the Experimental Section. The
peptides containing a biotin and a pentaglycine spacer at the amino-terminal end
were incubated with the metal ions before addition to the microtiter plate. The
monoclonal antibody specific for the N-terminal peptide was allowed to adsorb to
the surface of the plate, followed by the addition of the peptide-metal ion
complexes and the anti-biotin antibody conjugated with horseradish peroxidase. In
comparison to the indirect ELISA in which the antigen was immobilized through
Figure 20: HPLC profile a) and MALDI-FT-ICR mass spectrum b) of Biotin-(G)5-Aß(1-16).
0 10 20 30 40 50 60
0,2
0,4
0,6
0,8
1,0
1,2
1,4
A
bs (
22
0
nm )
Time (min)
21.9
1000 1500 2000 2500 3000 3500 m/z
2465.0861
A
bs (
22
0
nm )
Results and discussion 37
the biotin to the streptavidin coated plates, the sandwich ELISA provides the
advantage of allowing the interaction of the antibody with the free peptide-metal
ion complex thus minimizing the washing steps following addition of peptide-metal
ion complex.
In a first set of experiments, biotin-G5-Aß(1-16) and biotin-G5-Aß(1-10) were tested
for the binding to 6E10 and Bam-10. Both peptides reacted in a dose-dependent
manner with the 6E10 and Bam-10 antibodies (Figure 21). However, biotin-G5Aß(1-10) gave identical responses with both antibodies, while in the case of biotinG5-Aß(1-16) the affinity with the Bam-10 antibody was twofold higher than with
6E10.
a) b) Figure 21: Dose-response ELISA signal of biotin-G5-Aß(1-16) (squares) and biotin-G5-Aß(1-10)
(triangles) in the absence of transition metal cations, as measured with a) 6E10 (filled
symbols) and b) Bam-10 (open symbols).
0,0
0,2
0,4
0,6
0,8
1,0
0,001 0,01 0,1 1 10 100
ab so rb an ce (4
50
n m )
0,0
0,2
0,4
0,6
0,8
1,0
0,001 0,01 0,1 1 10 100
peptide concentration (µM)
biotin-G5-Aß(1-16)
biotin-G5-Aß(1-10)
biotin-G5-Aß(1-16)
biotin-G5-Aß(1-10)
ab so rb an ce (4
50
n m )
Results and discussion 38
In a further set of experiments the effect of different transition metal ions on Aß
recognition was tested. Different peptide dilutions (0.01-60 µM) and a fixed
concentration of metal ions (either 0 or 100 µM) (Figure 22) were employed in a
first experiment. The presence of 100 µM Zn2+ ions caused a significant increase
of the binding of biotin-G5-Aß(1-16) to both the 6E10 and Bam-10 antibody, which
resulted in a 4- and 10-fold increase in the ELISA response for 60 µM of Aßpeptide respectively. By contrast, the presence of Zn2+ did not influence the ELISA
response of biotin-G5-Aß(1-10). The cations Co
2+ and Ni2+ had no effect on biotinG5-Aß(1-16) recognition. The presence of Cu
2+ ions did not influence the
recognition of biotin-G5-Aß(1-16) by 6E10 mAb but resulted in a higher ELISA
response with the Bam-10 antibody.
a) b) Peptide concentration (µM)
Figure 22: Dose-response ELISA signal of biotin-G5-Aß(1-16) in the absence (open squares) or in
the presence of 100 µM Zn2+ (filled squares), Cu2+ (cross), Co2+ (filled triangles) or Ni2+
(filled lozenges) ions, as measured with 6E10 and Bam-10 mAbs.
0,0
0,5
1,0
1,5
2,0
A
b s o rb an ce (
45
0n m )
0,0
0,5
1,0
1,5
2,0
0,01 0,1 1 10 100
Biotin-G5-Aß(1-16)
Biotin-G5-Aß(1-16) + ZnCl2
Biotin-G5-Aß(1-16) + CuCl2
Biotin-G5-Aß(1-16) + CoCl2
Biotin-G5-Aß(1-16) + NiCl2
A
b s o rb an ce (
45
0n m )
Results and discussion 39
a) b) A further set of experiments was performed at a fixed peptide concentration (60
µM) but at increasing the metal ion concentration from 0 to 1.2 mM (Figure 23).
These experiments also showed a significant increase of biotin-G5-Aß(1-16)
Figure 23: Effect of divalent ions concentration on the ELISA response of biotin-G5-Aß(1-16)
(filled symbols) and biotin-G5-(1-10) (open squares),as measured with 6E10 and Bam10 mAbs. 60 µM of peptide were introduced in the presence of the indicated
concentrations of Zn2+ (squares), Cu2+ (cross), Co2+ (triangles) or Ni2+ (lozenges).
Biotin-G5-Aß(1-10) was tested only in the presence of Zn
2+.
0,0
0,5
1,0
1,5
2,0
A
b so rb an ce (
45
0n m )
0,0
0,5
1,0
1,5
2,0
0 100 200 300 400 500 600 700 800 900 1000 1100 1200
ion concentration (µM)
Biotin-G5-Aß(1-10)
Biotin-G5-Aß(1-16) + ZnCl2
Biotin-G5-Aß(1-16) + CuCl2
Biotin-G5-Aß(1-16) + CoCl2
Biotin-G5-Aß(1-16) + NiCl2
A
b so rb an ce (
45
0n m )
Results and discussion 40
recognition by the 6E10 and Bam-10 antibodies in the presence of zinc ions, which
was concentration dependant up to a plateau reached at 500 and 150 µM ZnCl2
for 6E10 and Bam10 respectively. As described above the presence of Zn2+ ions
had no effect on the recognition of biotin-G5-Aß(1-10). Likewise, the presence of
Co2+ or Ni2+ had no significant influence on the recognition of biotin-G5-Aß(1-16).
Consistent with the results obtained in the previous set of ELISA experiments,
Cu2+ ions at low concentration (up to 100 µM) led to an increased recognition of
biotin-G5-Aß(1-16) by the Bam 10 antibody, but had no effect on the recognition by
the 6E10 antibody.
From these results, it can be concluded that the sequence Aß(1-16) undergoes a
conformational change induced by the cation binding which results in an enhanced
recognition of the epitope by both antibodies. This result is consistent with the
three-dimensional structures of the Aß(1-16) (available at the pdb accession
number 1ze7) and Aß(1-16)-Zn2+ complex (available at the pdb accession number
1ze9). The three-dimensional structures were determined from the NMR spectra
recorded in aqueous solution, pH 6.5. According to these data, the first 6 amino
acids are unstructured while the region 7 to 15 of the Aß(1-16) has a well defined
structure but not canonical structure (see Figure 24). Upon zinc binding, the mainly
affected region of Aß(1-16) is (9-15), which contains three of the zinc ligand
residues Glu-11, His-13 and His-14.
Results and discussion 41
Figure 24: Three-dimensional structure of Aß(1-16) (a and b) and Aß(1-16) in complex with Zn2+
(c and d). The α-helix is depicted in red and the nonregular secondary structure is
indicated by green spline curves. The amino acids that are part of the zinc binding
domain are highlighted in the figures a and c and the residues 4-10 that are targeted
by the monoclonal antibodies are highlighted in the figures b and d.
In contrast, biotin-G5-Aß(1-10), which lacks the Glu-11, His-13 and His-14 residues
does not undergo a conformational change upon addition of Zn2+ and thus no
enhancement of the ELISA response is determined. Consequently this peptide
may be considered a negative control for the binding to the antibody in the
presence of divalent ions.
1
16
His6
His14
His13
Glu11
Zn 1
16
Phe4
Arg5 His6
Asp7
Ser8 Gly9
Tyr10Zn
His6 Glu11
His13
His14
1
16
Phe4
Arg5
His6
Asp7
Ser8
Gly9
Tyr10
a) b)
c) d)
Results and discussion 42
2.2. Epitope elucidation of Aß-specific human antibodies
2.2.1. Therapeutic potential of amyloid-specific autoantibodies from
human serum
The first promising results suggesting the existence of circulating Aß-antibodies
that are specific for antigens in AD emanated from observations by Gaskin and
colleagues of autoantibodies to neurofibrillary tangles in 1987 [123] and of
autoantibodies reactive to ß-amyloid in 1993 [124]. The approach employed in
these studies was to establish immortal cell lines by Epstein-Barr viral (EBV)
transformation of B cells from patients diagnosed with Alzheimer´s Disease, and to
test the supernatants containing soluble IgM molecules for the reactivity with Aß or
with amyloid plaques.
In a first study, the authors screened more than 6000 EBV-transformed cell lines
from six healthy individuals and six AD patients for the reactivity against AD brain
sections, and found only four cell lines able to secrete antibodies reactive against
plaques and blood vessels from AD brains [124]. All four antibodies were found to
be reactive with Aß(1-40) and Aß(1-28) but not with Aß(1-16) and Aß(12-28)
leading to the conclusion that there might be a contribution to the reactive epitope
by the sequence [29-40]. The antibodies were negative to the interaction with
myosin, actin, keratin, glial fibrillary acidic protein, tubulin and multiple antigenic
proteins. In a second study, the authors tested the supernatants of more than
3300 EBV-transformed B cell lines from thirteen individuals for the presence of
antibodies to Aß(1-40) by ELISA. Forty cell lines were identified to produce IgM
molecules reactive with Aß(1-40), however, the antibodies were plaque-unreactive
and apparently recognized different epitopes within the sequence of Aß. In
contrast to the plaque-reactive antibodies identified in the first study, that were
considered “monoreactive”, only 10 out of the 40 plaque-unreactive antibodies did
not react with any of the proteins tested, and 30 reacted with one or two of the
following proteins: keratin, actin, tubulin, myosin [125].
In a further study performed by Du et al., anti-Aß antibodies were detected in CSF
using an ELISA capture assay. The mean level of the anti-Aß antibodies in the AD
Results and discussion 43
group was significantly lower than in the control group. Significant and comparable
reduction of the binding to Aß was observed by ELISA if the CSF samples were
allowed to react prior to the assay with Aß(1-40), Aß(1-42) and Aß(25-35) [126].
Significantly lower titers of serum anti-Aß42 antibodies detected in AD patients
compared to elderly control individuals were also reported by another group [127].
In contrast, the studies performed by Hyman and collaborators [128] and Baril and
coworkers [129] showed low levels of anti-amyloid antibodies and no difference of
mean antibody levels between the healthy individuals and the AD patients.
The first report concerning a therapeutic potential of Aß-autoantibodies was
provided by Dodel et al. who showed that administration of intravenous gamma
globulins to patients with different neurological diseases reduced significantly the
total Aß and Aß(1-42) in CSF while the level of total Aß was found increased in
serum without significant change in Aß(1-42) [130]. Intravenous IgG preparations
are produced from plasma pools of healthy donors and are usually administered to
individuals with immune deficiencies. Anti-Aß antibodies isolated from purified
human plasma IgG by affinity media containing immobilized Aß(1-40) were able to
block fibril formation in vitro and to abolish the neurotoxicity exerted by both Aß(140) and Aß(25-35) towards cultured neurons [131]. A pilot study in which
intravenous immunoglobulin was administered to five AD patients showed a
reduction of total Aß concentration in CSF, whereas the serum levels of total Aß
increased [132]. Although an effect of the treatment on the cognitive function of the
patients was also investigated, no definite conclusions concerning the mental state
of the patients were reached from this pilot study. These studies focused on
autoantibodies against monomeric Aß. Recently O´Nuallain and coworkers found
that human sera as well as the immune globulin preparations derived from large
pools of plasma contain IgG molecules that recognize ß-amyloid peptide fibrils
[133]. The fibril-reactive antibodies were isolated by affinity chromatography using
fibril-conjugated Sepharose media and were shown to recognize cerebral amyloid
plaques and to inhibit fibrillogenesis.
Considering these previous data, human Aß-antibodies occurring in plasma and
CSF might be considered as a potential approach in the treatment of Alzheimer´s
disease. To pursue this hypothesis on a molecular level, the present study aims at
Results and discussion 44
the elucidation of the epitope(s) on Aß(1-40) recognized by autoantibodies, and to
correlate the results obtained for antibodies purified from pooled gamma globulin
preparations and from the serum of Alzheimer´s disease patients.
2.2.2. Purification of Aß-autoantibodies from pooled immunoglobulin
gamma preparations
Immunoglobulin gamma preparations are produced from large pools of human
plasma. Two different preparations were employed in the present work as source
of IgG for the purification of Aß-autoantibodies: (i), the immune globulin
intravenous (human) IGIV, produced by OCTAGAM is a liquid preparation of
highly purified IgG. The preparation contains 50 mg/ml IgG, 100 mg/ml maltose, ≤
5 µg/ml Triton-X100, ≤ 1 µg/ml tri-n-butyl phosphate (TNBP), IgA ≤ 0.1 µg/ml, IgM
≤ 0.1 µg/ml. 96% of the IgG is human normal immunoglobulin containing ≤ 3%
aggregates, ≤ 3% fragments, 90% monomers and dimmers with a final sodium
content of 30 mM and a pH of 5-6. This product is administered intravenously to
individuals with immune deficiencies. (ii), A second IgG preparation used, was
human gamma-globulin from Calbiochem. This product is a lyophilized solid
containing 97% IgG, produced only for research use without clinical use.
To purify the specific anti-Aß antibodies from the serum IgG preparations an
affinity column containing immobilized Cys-Aß(1-40) was used. Due to the
presence of 2 lysine residues within the sequence of Aß(1-40) the immobilization
of the peptide to the N-hydroxysuccinimide activated Sepharose based on the
primary amino groups was not possible. The Ultralink® Iodoacetyl Gel reacts
specifically with free sulfhydryls to form a stable thioether linkage. Since Aß(1-40)
contains no cysteine residue, a peptide derivative containing an aditional Nterminal cysteine residue was synthesized to provide the covalent immobilization
of the peptide to the matrix by thioether coupling (Figure 25).
Figure 25: Schematic representation of affinity ligand coupling by thioether bond formation.
S-CH2-CH-CO-Aß(1-40)
N
H
O
I + HS-CH2-CH-CO
NH2
- HI
N
H
O
NH2
-Aß(1-40)
Results and discussion 45
For the purification of Aß-autoantibodies from immune globulin preparations, 250
mg of human IgG were diluted in PBS and incubated with the Cys-Aß(1-40) affinity
column, for 12 hours at 4°C. After washing the bound antibodies were eluted
under acidic conditions. The detailed procedure is described in the experimental
part and depicted schematically in Figure 26. Passage of the immunoglobulin
solution through the column yielded two fractions: the first one consisted of
unbound IgG present in the PBS filtrate and a second of Aß-bound antibodies
eluted by the acidic treatment. The concentration of IgG in the eluted fraction was
determined by the MicroBCATM Protein Assay Kit. Based on the immunoglobulin
content of the IgG solution subjected to the affinity chromatography and of the
eluate, the concentration of Aß-antibodies within the human immune globulin
preparation was determined. An example of the Aß-autoantibody separation will
be given in par. 2.2.3. for the immune globulin preparation purchased from
Calbiochem.
Figure 26: Schematic representation of the antibody purification using immobilized antigen column.
The IgG preparation is incubated overnight with the affinity media containing
immobilized Aß(1-40) followed by removal of unreacted IgG molecules and acid-elution
of Aß-specific antibodies.
Results and discussion 46
2.2.3. Identification of the epitope recognized by Aß-autoantibodies
isolated from pooled immunoglobulin gamma
The Aß-specific autoantibodies present in IVIG (OCTAGAM) were purified on
affinity matrix containing immobilized Cys-Aß(1-40). The integrity of the purified
Aß-autoantibodies was assessed by separation of the components present in the
sample on a 1D-gel performed under denaturing and reducing conditions. The 1-D
gel separation of the Aß-autoantibody sample is shown in Figure 27. The intense
spots of the heavy and light chain indicate that the antibody contained >90 %
intact antibody chains.
The identification of the epitope recognized by the Aß-reactive antibody from IVIG
was performed using an affinity column containing 100 µg of immobilized antibody
(column prepared according to the procedure described in the Experimental
Section). The reactivity of the affinity column to Aß and the optimal amount of
buffer for the removal of non-specifically bound Aß were tested in a first
experiment. 25 µg of intact Aß was dissolved in PBS to a final concentration of 0.1
µg/µl and allowed to react with the antibody for 2 hours. The supernatant
containing excess peptide was then removed and the column was washed with 30
ml of PBS. The last volume of the washing buffer was collected for mass
spectrometric analysis in order to investigate whether non-specifically bound
peptide fragments were present. The affinity bound peptides were then dissociated
Figure 27: 1-D gel of human Aßautoantibody purified from IVIG and
visualized by colloidal Coomassie.
67
45
36
29
24
20
14
Marker
ProteinsKDa
Aß-autoantibody
Heavy chain
Light chain
Results and discussion 47
by treatment with 0.1% trifluoroacetic acid. Figure 28 shows the MALDI-MS
analyses of the supernatant, washing and elution fractions. Molecular ions
corresponding to Aß(1-40) ([M+H]+calc. of 4328.88) were identified in the
supernatant and elution fraction. This experiment confirmed that the column
containing immobilized Aß-antibody was able to react with Aß(1-40) and the nonspecifically bound fragments can be removed by washing with 30 ml of PBS.
a) b) c) For the mass spectrometric identification of the epitope, a series of proteolytic
cleavages were performed to remove the Aß-residues that were unprotected by
the autoantibody binding. Endoproteinase GluC from Staphylococcus aureus
strain V8 is able to cleave peptide bonds at the carboxyl side of glutamyl residues
Figure 28: MALDI-TOF mass spectra of the Aß affinity experiment with the immobilized Aßautoantibodies. a) Supernatant, b) Wash test, c) Elution fraction.
[M+H]+ calc.= 4329.8
Aß(1-40)
Aß(1-40)
[M+2H]2+
Results and discussion 48
if the reaction is carried out in ammonium bicarbonate (pH 7.8) and provides
cleavage at the carboxyl side of either glutamyl and aspartyl residues if the
reaction is carried out in phosphate buffer (pH 7.8). Due to the presence of 3
aspartyl residues and 3 glutamyl residues within the Aß(1-40) sequence the
proteolytic cleavage was performed in phosphate buffer. Digestion of Aß(1-40) by
GluC for 4 hours resulted in cleavage at the Glu-3 and Glu-11 residues. The
peptide mixture produced by enzymatic proteolysis using GluC was allowed to
react with the affinity column for 2 hours. MALDI-MS analysis of the unbound
peptide fragments revealed the fragments [4-11], [1-11] and [12-40] whereas the
mass spectrum of the elution fraction contained an abundant ion of m/z 3022.2
that corresponded in mass to the amino acid sequence [12-40]. In order to verify
whether a shorter sequence than [12-40] is able to display binding to the antibody,
Aß(1-40) was digested in solution for 20 hrs and the resulting peptide mixture was
exposed to the affinity column. The supernatant contained peptide fragments
resulting from cleavage at Glu-3, Asp-7, Glu-11, Glu-22 and Asp-23. The peptide
fragments identified in the supernatant and elution fraction are shown in the Figure
29 a and b and summarized in Table 16 (par. 3.6.3.).
Although longer N-terminal peptide fragments such as [1-11], [12-22] and [1-23],
were produced at extended digestion time (20 hrs) with GluC, the elution fraction
contained only the Aß(12-40). This result showed that the epitope is not contained
in the N-terminal sequence [1-11] and in the sequence [12-22]. However, the
carboxy-terminal fragments [23-40] and [24-40] present in the supernatant did not
show affinity to the autoantibody. These preliminary data were interpreted to
suggest that the autoantibody might recognize a middle domain of Aß. Consistent
with the results provided by epitope extraction, the predominant peptides identified
in the mass spectrum of the elution fraction upon epitope excision performed by
digestion of immobilized Aß(1-40) with GluC for 20 hrs, were [12-40] and intact
Aß(1-40) (see Figure 29c).
Results and discussion 49
a) b) A second enzyme employed for proteolytic excision was trypsin. Aß(1-40) contains
3 possible cleavage sites of trypsin: Arg-5, Lys-16 and Lys-28. Considering the
sequence Aß(12-40) identified by proteolytic digestion with endoproteinase GluC,
the proteolytic reaction at the Lys-17 and Lys-28 residues could provide additional
information. Upon cleavage by trypsin of Aß(1-40) in complex with the antibody,
the mass spectrum of the elution fraction contained an intense ion at m/z 3707.9
due to the tryptic peptide [6-40] [M+H]+calc.= 3711.2 and with lower abundance an
ion at m/z 2391.5 corresponding to the fragment [17-40] [M+H]+calc.= 2392.8 (see
Figure 29: MALDI-TOF mass spectra of the epitope identification using GluC a) the supernatant
of the epitope extraction b) the elution fraction after epitope extraction c) elution
fraction obtained by epitope excision with endoproteinase GluC The insert in the upper
right corner of the figure contains the Aß(1-40) sequence. The possible cleavage sites
of GluC are indicated by a square and the cleavage sites observed by the
identification of the proteolytic peptides present in the supernatant are indicated by
arrows.
Aß(4-11) Aß(1-11)
Aß(12-40)
Aß(1-40)
Aß(4-7)
Aß(12-22)
Aß(24-40)
Aß(23-40)
Aß(1-23)
Aß(4-22)
Aß(12-40)
Aß(1-40)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
Aß(12-40)
Aß(1-40)
c) Results and discussion 50
Figure 30). The ions at m/z 637.5 and 1335.4 observed in the supernatant were
identified as singly charged ions of the fragments Aß(1-5) [M+H]+calc.= 637.6 and
Aß(6-16) [M+H]+calc.= 1337.3. The low intensity of the molecular ion corresponding
to the peptide Aß(1-17) might indicate that the cleavage at Lys16 residue by
trypsin occurs with a lower rate, and possibly suggest that the sequence around
this residue might be involved in the binding to the antibody.
a) b) In order to probe more possible cleavage sites within the sequence, the epitope
excision experiment was also carried out using α-chymotrypsin as protease which
catalyzes the hydrolysis of peptide bonds at the C-terminal side of Phe, Tyr, Trp
and Leu peptide bonds. Hydrolysis of peptide bond at slow rates also occurs at
Figure 30: MALDI-TOF mass spectra showing the ions observed in the supernatant a) and
elution fraction b) produced by epitope excision with trypsin. The insert shows the
sequence of Aß(1-40). The possible cleavage sites are indicated with squares and
the cleavage sites determined upon identification of the tryptic fragments present in
the supernatant and elution fractions are indicated with arrows.
Aß(1-5) Aß(6-16)
Matrix
Aß(1-40)
Aß(6-40)
Aß(17-40)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
Results and discussion 51
Met, Ile, Ser, Thr, Val, His, Gly and Ala. The mass spectrum (Figure 31 a)
acquired for the peptide mixture resulting from the digestion of Aß(1-40) by alphachymotrypsin for 20 hrs revealed several ions that are summarized in Table 18
(par. 3.6.4.) The digestion led to proteolytic cleavage at all the primary cleavage
sites, Tyr-10, Phe-4, Phe-19, Phe-20 and Leu-17 and one of the secondary
cleavage sites Met-35, whereas the digestion of Aß(1-40) in complex with the
antibody led to cleavage only at the Phe-4 residue. Thus, the mass spectrometric
analysis of the elution fraction provided the identification of fragment Aß(5-40)
while the supernatant contained no signal.
a) b) To examine if the amino acid sequence of the epitope can be further reduced,
epitope excision was carried out using pronase. In contrast to the proteases
Aß(5-40)
Aß(5-10)
Aß(28-35)
Aß(18-27)
Aß(11-17)
Aß(21-35)
Aß(5-17) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1 40
Figure 31: MALDI-TOF mass spectra of the peptide mixture produced by proteolytic digestion of
Aß(1-40) by alpha-chymotrypsin in solution a) and the elution fraction upon epitope
excision using alpha-chymotrypsin b). The insert shows the Aß(1-40) sequence. The
proteolytic cleavage sites identified by digestion of Aß(1-40) in solution are indicated
by arrows. The peptide eluted after epitope excision using alpha-chymotrypsin is
highlighted in grey.
Results and discussion 52
employed in the previous experiments, pronase is a non-specific protease able to
cleave proteins down into individual amino acids and exerts its proteolytic activity
on denatured as well as on native proteins. This property is attributed to the
composition of the preparation, comprising several types of endoproteinases
(serine and metaloproteases) and exoproteinases (carboxypeptidases and
aminopeptidases). The optimum pH value is 7.0-8.0 and the optimum temperature
is in the range 40-60ºC. Aß after complexing with the antibody was treated with
Pronase (0.5 µg/µl) for 2 hrs at 40ºC, and compared to the digestion in solution.
No molecular ion could be identified in the mass spectrum acquired for the
digestion mixture of Aß in solution after 2 hours, which indicated pronase
degraded free Aß to the individual amino acids (see Figure 32).
a) b) The mass spectrum of the elution fraction collected upon epitope excision with
pronase provided the identification of several Aß(1-40) fragments as summarized
in the Table 2. The proteolytic cleavage by pronase of Aß(1-40) in complex with
the autoantibodies occurred predominately at the N-teminal part of the peptide. All
the fragments resulted were truncated at the N-terminal part and one fragment,
Figure 32: MALDI-TOF mass spectra of a) Aß(1-40) digested in solution with Pronase for 2 hours
and b) Aß(1-40) peptide fragments eluted after epitope excision using Pronase.
Aß(21-40)
Aß(14-40) Aß(7-40)
Aß(11-37)
Aß(6-40)
Aß(5-40)
Aß(1-40)
Results and discussion 53
Aß(11-37) was identified upon cleavage at both N-and C-terminal ends of the Aßsequence. The cleavages at the amino-terminal side occurred after amino acid
residues Phe-4, Arg-5, His-6, Tyr-10, His-14 and Phe-20. The only C-terminal
cleavage occurred after Val-36. Considering these results, the minimal sequence
that preserves the affinity to the antibody was [21-37]. The presence of an
additional molecular ion containing methionine sulphoxide for each of the
sequences [11-37], [7-40], [6-40], [5-40] and [1-40] confirmed the correct
identification as the Aß-sequence contains only one methionine residue located in
the C-terminal part (Met-35). The ions containing oxidized methionine are
indicated in the mass spectrum by an arrow.
Table 2: Peptide sequence assignment of the molecular ions present in the elution fraction after
digestion of the immune complex with pronase
[M+H]+exp [M+H]
+
calc. ∆m
Da Proteolytic
fragment
Sequence
1885.5 1886.22 0.72 [21-40] 21AEDVGSNKGAIIGLMVGGVV40
2786.3 2786.31 0.01 [14-40] 14HQKLVFFAEDVGSNKGAIIGLMVGGVV40
2895.3 2897.36 2.07 [11-37] 12EVHHQKLVFFAEDVGSNKGAIIGLMVG37
3572.4 3574.09 1.70 [7-40] 7DSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
3709.8 3711.23 1.44 [6-40] 6HDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
3866.5 3867.42 0.99 [5-40] 5RHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
The various proteolytic enzymes provided detailed information about the sequence
recognized by the Aß-autoantibodies (see Figure 33). The minimal sequence [2137] was determined by epitope excision using pronase. However, the information
provided by the specific enzymes (trypsin, GluC and α-chymotrypsin) significantly
contributes to the unequivocal identification of the epitope. Proteolytic cleavages of
Lys-28 by trypsin, Glu-22 and Asp-23 by GluC were not observed confirming that
these residues are shielded in the immune complex. Noteworthy, the Lys-17
residue is cleaved at a very low rate indicating that the activity of trypsin at this site
might be sterically hindered. Similarly, the epitope excision with alphachymotrypsin reveals that the enzyme is able to hydrolyze only the peptide bond
following Phe-4, and cleavage after Tyr-10, Leu-17, Phe-19 and Phe-20 are
hindered when Aß is bound to the antibody while in the free form the enzyme is
able to hydrolyze these peptide bonds.
Results and discussion 54
These results ascertained that the Aß-antibody isolated from the IVIG preparation
recognizes an epitope located in the carboxy-terminal part of Aß within the amino
acid residues [21-37]. Presumably due to the polyclonality of the antibody amino
acid residues located in the sequence [12-20] might also contribute to an
increased reactivity of the Aß to the autoantibody.
In contrast to the IVIG the human IgG preparation from Calbiochem is a lyophilized
solid prepared only for research use. In a further part of study it was investigated
whether this product contains anti-Aß autoantibodies and the epitope specificity
was compared with the Aß-autoantibodies isolated from IVIG. 250 mg of the solid
powder containing IgG were reconstituted in PBS and incubated with the CysAß(1-40) affinity column according to the procedure detailed in the Experimental
Part. Based on the amount of specific Aß-autoantibody quantified in the eluate the
Aß-antibodies represented 0.1-0.2% of the IgG molecules in the Calbiochem
product. In order to identify the epitope recognized, an affinity column containing
immobilized Aß-antibodies was prepared. 150 µg of the Aß-antibody quantified in
the elution fractions of the antigen column were lyophilized and reconstituted in
coupling buffer to a final concentration of 0.5 µg/µl. The solution was applied to
Sepharose matrix and the antibody was covalently immobilized according to the
procedure described in the Experimental Part. To ensure minimal non-specific
interaction of the antigen to the column material, the amount of antigen applied to
the antibody column was reduced to 5 µg of intact or digested Aß(1-40). Due to
the identification in the previous epitope identification experiments of the sequence
[12-40] and [21-37], the enzyme of choice was GluC protease, at a digestion time
of 20 hours.
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
1
GluC
Pronase
Trypsin
Figure 33: Summary of the results leading to the identification of the Aß(21-37) epitope. The
cleavages that occur when the antigen-autantibody complex is subjected to
enzymatic proteolysis are indicated by continuous arrow lines and the shielded
cleavage sites by dashed arrow lines.
Results and discussion 55
The MALDI-TOF mass spectra of the supernatant and elution fractions from the
epitope extraction are shown in the Appendix 3, Figure 83. In the mass spectrum
of the supernatant, ions corresponding to the N-terminal peptide [4-11], to the
middle domain [12-22], to fragments containing missed cleavage sites [12-40], [440] and intact [1-40] were identified. The elution fraction contained only N-terminal
truncated peptides with the most intense ion signal corresponding to the sequence
[12- 40]. In the case of the epitope excision, a low amount of Aß(1-40) remained
unbound and was identified in the mass spectrum of the non-binding fraction. The
supernatant, collected after incubation for 20 hrs with GluC contained Aß(1-40)
and the fragment [12-22] (see Figure 84). After washing the column with 30 ml
PBS the last volume of the PBS was collected to check by mass spectrometry for
background ions prior to elution. The mass spectrum of the washing fraction
yielded no contaminant ion while the mass spectrum of the elution fraction
contained molecular ions corresponding to the fagments [12-40] and [4-40] with
low intensity and the intact Aß(1-40) with high intensity. The difference in the
intensities of the fragment [12-40] and the intact Aß(1-40) that can be observed in
the elution fractions of the epitope extraction and epitope excision suggest a lower
rate of the proteolytic cleavage of Aß(1-40) by GluC in the complexed state.
In a further set of experiments an affinity column containing unfractioned IgG was
prepared using identical experimental conditions as for the immobilisation of the
Aß-antibody. Considering that the concentration of Aß-autoantibody is
approximately 0.1-0.2% of the total IgG, 150 µg of IgG should contain maximum
0.3 µg of Aß-autoantibody which is negligible for showing a specific interaction
with Aß. Therefore the column containing 150 µg of immobilized IgG represents a
suitable control to examine the unspecific affinity that might appear during the
epitope identification due to the interaction of intact Aß(1-40) and of the proteolytic
fragments with the matrix, the column or with domains of the IgG that are not part
of the CDRs. The epitope extraction using the unfractionated IgG control column
was carried out in parallel with the experiments performed on the Aß-antibody
column. The mass spectra of the supernantant collected after exposing the
proteolytic mixture resulting from digestion of Aß with GluC to the column showed
molecular ions corresponding to the fragments [4-11], [12-22] and [12-40]. The
elution fraction from the epitope extraction on the control column showed the
Results and discussion 56
absence of any molecular ion (Figure 85). The epitope excision on the
unfractioned IgG column was carried out in the same manner in comparison to the
epitope excision on the Aß-autoantibody column. The non-binding fraction
collected after 2 hrs of incubation with the IgG column contained unbound Aß(1-40)
(Figure 86). The enzyme was added to the column and incubated for 20 hrs
followed by removal of the supernatant fraction. The mass spectrometric analysis
revealed the presence of Aß(1-40) at a very low extent. The elution fraction
contained a molecular ion corresponding to Aß(1-40).
The epitope identification carried out for the Aß-autoantibody purified from the
immune globulin preparation purchased from Calbiochem showed that the epitope
resides within the sequence [12-40] and is consistent with the epitope recognized
by the Aß-autoantibodies purified from IVIG. The control experiments carried out
for the examination of the non-specific binding of the Aß(1-40) peptides indicated
that intact Aß(1-40) has nonspecific binding to the affinity column, however the
molecular ion pattern identified in the elution fraction of the epitope identification
using the autoantibody affinity column was not observed at the control
experiments.
2.2.4. Clonal diversity and sequence analysis of human serum Aß
antibodies by 2D-gel electrophoresis and peptide mass fingerprint
The sequence identification of an unknown antibody molecule involves the use of
several methods (see Figure 34). After affinity isolation of the Aß-autoantibodies
from the immunoglobulin gamma preparation, the protein was reduced with DTT
and the sample, containing monomers of heavy and light chains is subjected either
to 2D-electrophoresis (method A) or to 1D-Gel electrophoresis (method B). In the
method A the antibody molecules are separated according to the isoelectric point
and the molecular weight. The spots identified in the gel were digested by trypsin,
and eluted from the gel pieces in order to be analysed by MALDI-FTICR-MS. This
method is able to provide identification of sequences that are part of the constant
chains. The peptides that derive from variable domains and hypervariable domains
of the immunoglobulins can be identified with this method only if the sequence is
Results and discussion 57
included in the protein database. This approach was employed in the present work
for the analysis of the clonal diversity of human serum Aß-autoantibodies.
Figure 34: Analytical approach antibody sequence analysis.
In the method B the monomers were separated according to the molecular weight
in heavy and light chains. Each spot contains a mixture of either heavy or light
chains deriving from different antibody clones. The spots are in-gel digested and
the peptide fragments are analysed by LC-MS/MS whereby primary structure
information can be obtained for every peptide separated by collision-induced
dissociation. Alternatively, the peptides present in the digestion mixture can be
separated by HPLC followed by either MALDI-TOF-MS or Edman sequencing of
the individual peptides.
The 2-DE has been shown to be particularly useful to resolve complex mixtures of
proteins as well as to study the clonality of the IgGs. With this technique
monoclonal chains are differentiated from polyclonal chains according to their 2-D
patterns. Polyclonal heavy and light chains are highly heterogeneous and are
resolved as diffuse zones, whereas monoclonal chains show charge and to a
lesser degree, size microheterogeneity [134-136]. The biochemical origin of the
microheterogeneity of monoclonal heavy and light chain is mainly expected to
Affinity Isolation of anti-Aß autoantibodies
Reduction with DTT
In-gel Digestion
A B2D-Gel
Electrophoresis
MALDI-FTICR-MS
1D-Gel
Electrophoresis
Constant and Var.
Region Peptides
In-gel Digestion
HPLC LC-MS/MS
Constant and Var.
Region Peptides
MALDI-TOF-MS
Edman
sequencing
Constant and Var.
Region Peptides
Constant
Region Peptides
Results and discussion 58
derive from post-translational modifications but this hypothesis has not been yet
ascertained. The panels a) and b) of the Figure 35 show the 2D-gel separation of
the purified Aß-reactive antibody and of the commercial polyclonal anti-lysozyme
antibody respectively.
a) b)
The IgG chain patterns of the Aß-autoantibody emerge in a large region of the 2D
gels throughout most of the pI range 6 to 10 for both the heavy chains and the light
chains. The resulting 2D patterns were compared to those obtained with a
commercially available polyclonal anti-lysozyme antibody. Similar to the Aßautoantibody the heavy chains appear throughout the pI range 6 to 10 and show a
high degree of charge heterogeneity whereas the light chains are visible in the pI
range 5 to 6.5 and display both charge and molecular weight heterogeneity.
The heavy chain spots 4, 12 and 13 (see Figure 36) were excised, destained and
the protein was subjected to enzymatic proteolysis by trypsin according to the
method outlined in the Experimental Part.
Figure 35: 2D-SDS-PAGE comparison of the molecular heterogeneity of a) Aß-autoantibody
purified from IVIG visualized by Coomassie Blue staining and b) Polyclonal antilysozyme antibody visualized by silver staining.
6 7 8 94 5pI 6 7 8 94 5pI
Heavy chain
Light chain
Heavy chain
Light chain
Results and discussion 59
The molecular masses of each set of peptides were determined by MALDI-FT-ICR.
The mass spectrum of the tryptic peptides obtained from the protein visualized in
spot 12 is shown in the Figure 37. The set of molecular weights experimentally
assessed were used to search the mass profiles generated by theoretical
fragmentation of the proteins included in the NCBInr database. The database
interrogation was performed with the MASCOT search engine by selecting a mass
tolerance of maximum 30 ppm and the carbamidomethyl permanent modification
of cysteine residues. The sequence assignment of the peptides identified from the
spots 4, 12 and 13 are shown in the Table 3.
Figure 36: Section of the 2D gel depicting the separation of the Aß-autoantibody heavy chain.
Polyclonal γ-chains appear as a cloudy zone without well distinguishable spots. The
spots 4, 12 and 13 were subjected to in-gel digestion followed by mass
spectrometric analysis of the proteolytic mixture produced.
[9
6-
10
6]
[3
72
-3
82
]
[1
49
-1
60
]
[3
88
-3
97
]
[3
16
-3
28
]
[3
02
-3
15
]
[3
29
-3
44
]
[2
83
-3
01
]
[3
98
-4
19
]
[4
44
-4
66
]
[2
50
-2
75
]
[2
46
-2
75
]
[9
6-
10
6]
[3
72
-3
82
]
[1
49
-1
60
]
[3
88
-3
97
]
[3
16
-3
28
]
[3
02
-3
15
]
[3
29
-3
44
]
[2
83
-3
01
]
[3
98
-4
19
]
[4
44
-4
66
]
[2
50
-2
75
]
[2
46
-2
75
]
Figure 37: Mass spectrum of the peptide mixture resulted by trypsin treatment of the spot 12.
Results and discussion 60
Table 3: Sequence assignment of the masses determined by MALDI-FT-ICR mass spectrometry
based on the correlation to tryptic fragments mass profiles of the proteins included in the
database.
[M+H]+exp [M+H]
+
calc. ∆m
in ppm
Spot
4
Spot
12
Spot
13
Residue
number
Sequence
1161.6406 1161.6296 9 + [380-389] NQVSLTCLVK
1186.6563 1186.6467 8 + [141-152] GPSVFPLAPSSK
1286.6858 1286.6739 9 + + + [364-374] EPQVYTLPPSR
1352.7132 1352.6991 10 + [95-105] NTLYLQMNSLR
1671.8411 1671.8085 20 + [308-320] TKPREEQYNSTYR
1677.8217 1677.8020 24 + + [294-307] FNWYVDGVEVHNAK
1808.0597 1908.0065 29 + + [321-336] VVSVLTVLHQDWLNGK
2139.0681 2139.0275 19 + + [275-293] TPEVTCVVVDVSHEDPEVK
2544.1605 2544.1314 11 + + + [390-411] GFYPSDIAVEWESNGQPENNYK
2801.3065 2801.2671 14 + + + [436-458] WQQGNVFSCSVMHEALHNHNTQK
2844.4838 2844.4576 9 + + [242-267]* THTCPPCPAPELLGGPSVFLFPPKPK
3334.6749 3334.6422 10 + [238-267] SCDKTHTCPPCPAPELLGGPSVFLFPPKPK
1882.0266 1882.0336 4 + [1-15] MMEFWLSWVFLVAILK
1873.0026 1872.9702 17 + [364-379] EPQVYTLPPSRDELTK
3036.5243 3036.4967 9 + [241-267]* KCCVECPPCPAPPVAGPSVFLFPPKPK
2908.4326 2908.4017 11 + [242-267]* CCVECPPCPAPPVAGPSVFLFPPKPK
1794.0128 1793.9909 12 + [321-336]* WVSVLTVVHQDWLNGK
+ The sign indicates an identical sequence identified in the different spots analyzed
* The asterisk indicates that the sequence contains amino acids that are responsible for the differentiation
between the subtypes gamma 1 and gamma 2 of the IgG
The MS data of the spot 12 provided partial identification (38%) of the heavy chain
constant region of the polyclonal IgG autoantibodies (Figure 38). Additional
information was provided by the identification of the amino acid sequence [1-15]
from the MS data of the spot 4. The amino acid residues [242-255] contained in
the tryptic fragment [242-267] are different in the case of the spot 13 if compared
with the result from both spots 12 and 4. These residues are located in the hinge
region known to display the highest degree of subtype variability and indicate that
the subtype of the immunoglobulin visualized in the spot 13 is gamma-2 while for
the spots 4 and 12 the subtype is gamma-1. From the 13 peptides identified only 2
were in the variable region of the heavy chain and the amino acid residues [101105] comprised by the tryptic sequence [95-105] are part of the CDR.
Results and discussion 61
In the case of the protein spots annotated for the light chain molecules of the Aßautoantibodies (Figure 39), the spot 4 was subjected to in gel digestion and
subsequent mass spectrometric analysis.
The molecular masses determined by MALDI-FT-ICR mass spectrometric analysis
of the tryptic mixture from the spot 4 (Figure 40) provided the identification of
kappa light chain. The peptide sequences were identified with an average mass
error of 4 ppm. The coverage of the kappa light chain provided by the identified
sequences is 43%. Within the amino acid residues of light chain only the sequence
[62-77] is part of the variable domain which comprises the residues [1-107],
whereas 77 % of the constant domain of the light chain [108-214] was covered.
Figure 39 Section of the 2D gel depicting the separation of the Aß-autoantibody light chain.
The charge heterogeneity of the light chains appears less pronounced in
comparison to the heavy chains. The spot 4 was subjected to in-gel digestion
followed by mass spectrometric analysis of the proteolytic mixture produced.
Figure 38: MASCOT browser output depicting the sequence coverage for the immunoglobulin gamma
heavy chain isotype 1 (IGHG1) based on the correlation of the MS spectrum provided by
the tryptic digest of the spot 12 to peptide sequences from the database. The residues
underlined are identified from the spot 4.
Results and discussion 62
Table 4: Sequence assignment of the masses determined by MALDI-FT-ICR mass spectrometry
based on the correlation to tryptic fragments mass profiles of the proteins included in the
database
[M+H]+exp [M+H]
+
calc. ∆m
in ppm
Spot
4
Residue
number
Sequence
1632.7917 1632.7864 3 + [62-77] FSGSGSGTDFTLTISR
2102.1382 2102.1280 5 + [108-126] RTVAAPSVFIFPPSDEQLK
1946.0343 1946.0269 4 + [109-126] TVAAPSVFIFPPSDEQLK
1797.9012 1797.8952 3 + [127-142] SGTASVVCLLNNFYPR
2135.9785 2135.9687 5 + [150-169] VDNALQSGNSQESVTEQDSK
2141.0916 2141.0808 5 + [189-207] HKVYACEVTHQGLSSPVTK
1875.9350 1875.9269 4 + [191-207] VYACEVTHQGLSSPVTK
[6
2-
77
]
[1
08
-1
26
]
[1
09
-1
26
]
[1
27
-1
42
]
[1
50
-1
69
]
[1
89
-2
07
]
[1
91
-2
07
]
[6
2-
77
]
[1
08
-1
26
]
[1
09
-1
26
]
[1
27
-1
42
]
[1
50
-1
69
]
[1
89
-2
07
]
[1
91
-2
07
]
Figure 40: Mass spectrum of the peptide mixture resulted by trypsin treatment of the spot 4.
Results and discussion 63
2.2.5. Identification of the epitope recognized by Aß-autoantibodies
isolated from the serum of an Alzheimer disease patient
In order to investigate whether the Aß-autoantibodies present in the blood of
Alzheimer Disease patients have the identical or different epitope specificity, an
affinity column was prepared using 100 µg of the Aß-autoantibody sample from an
AD patient (AD77). 5 µg of Aß-autoantibody were compared with the first 4
aliquots of the flow-through fraction obtained by immobilization of the antibodies
on Sepharose. All the samples were reduced with DTT and solubilised in sample
buffer and the proteins were separated by 1D-gel electrophoresis as described in
the section 3.4.3 (see Figure 41).
Figure 41: 1D-gel of human Aß-autoantibody AD77. The stock solution of Aß-autoantibody is
compared with the first 4 aliquots collected after immobilization on Sepharose.
2 spots were identified in the gel in the case of the Aß-autoantibody stock solution
corresponding to the molecular weight of the heavy and light chains. The 2 bands
that were present above the heavy and light chains indicate that the sample was
not completely denatured before the gel was run. The fade band that can be
distinguished in the wash 1 indicates that an amount of antibody that is lower than
5 µg remained unbound and more than 95% of the antibodies were immobilized on
the Sepharose.
67
45
36
29
24
20
AD77
Stock
solution
Wash 1 2 3 4Marker
Proteins
KDa
Heavy chain
IgG
Light chain
IgG
Results and discussion 64
Aß(1-40) was digested with GluC for 20 hrs and applied to the affinity column
containing 100 µg of Aß-autoantibody purified from the serum of the AD77 patient.
The Figure 42 shows the mass spectra of the supernatant and elution fractions.
The supernatant contains N-terminal fragments [1-11], [2-11], [4-11], C-terminal
fragments [23-40] and longer sequences [12-40], [4-40]. In the epitope fraction the
minimal sequence identified was [23-40]. Longer sequences displaying N-terminal
truncations are present in the elution fraction [4-40], [8-40], [12-40]. This epitope
identification indicated that the epitope resides within the amino acid residues [2340]. The result is consistent with the identification of the epitope [21-37] in the case
of the Aß-autoantibodies purified from the immunoglobulin gamma preparations
that are produced from pooled human plasma from healthy individuals.
Figure 42: MALDI-FT-ICR mass spectra of the epitope extraction using GluC a) supernatant b)
elution fraction.
Aß(4-11)
Aß(2-11)
Aß(1-11)
Aß(23-40)
Aß(12-40)
Aß(4-40) Aß(1-40)
Aß(1-40)
Aß(23-40)
Aß(8-40)
Aß(4-40)
Aß(12-40)
Aß(12-40)*
b) a) Results and discussion 65
Table 5: Sequence assignment of molecular ions observed in the elution fraction from the epitope
extraction using GluC
[M+H]+exp [M+H]
+
calc. ∆m
ppm
Proteolytic
fragment
Sequence
1684.9411 1684.9415 0 [23-40] 23VGSNKGAIIGLMVGGVV40
3020.6561 3020.6503 2 [12-40] 12VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
3036.6266 3036.6497 7 [12-40]* 12VHHQKLVFFAEDVGSNKGAIIGLM*VGGVV40
4012.0549 4012.0651 3 [4-40] 4FRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
4327.0248 4327.1717 49 [1-40] 1DAEFRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
* Oxidation at Met-35
2.2.6. Synthesis of biotinylated amyloid peptides encompassing the
epitope
Although solid phase peptide synthesis provides an established method for the
preparation of a wide range of peptide sequences, a number of peptide sequences
remain critical to prepare. Aß accounts to such so-called “difficult” sequences due
to the high hydrophobicity of the C-terminal segment resulting in a high tendency
to aggregate in solution as well as on-resin during the elongation of the peptide
chains [137, 138]. The Aß-peptides were synthesized by Fmoc chemistry using
TGR resin as support. In comparison to the N-terminal Aß- sequences, where the
Fmoc deprotection was achieved with 20% piperidine, in the case of the
sequences containing C-terminal fragments the deprotection was performed with a
solution containing 2% DBU, 2% piperidine in DMF [139, 140] using a deprotection
time of 7 minutes. To ensure the availability of fresh solvents the synthesis was
carried out in steps of 10 amino acid residues using a double coupling protocol
and a coupling time of 20 min. To confirm the synthesis of the desired sequence
after each step, the resin was washed with DMF and dried in the presence of
vacuum. An aliquot of resin was subjected to cleavage for 30 min and the peptides
released in the solution were applied on a sample target, mixed with HCCA
solution and analysed by mass spectrometry. After the buildup of the desired
amino acid chain the peptide was cleaved from the resin and precipitated as
described in the Experimental Part. The precipitate was resolubilised in
CH3CN/H2O/TFA (66:33:0.1v/v/v) due to the poor solubility in 5% acetic acid and
lyophilized. HPLC was performed using a preparative DELTA-PAK C4 column and
analytical RP-HPLC was carried out on a Vydac C4 column. The HPLC profile and
Results and discussion 66
the MALDI-FT-ICR mass spectra of the purified peptides are shown in the Figure
43.
a) b) c) Figure 43: Analytical RP-HPLC and MALDI-FT-ICR mass spectra of pure a) Biotin-(G)5-Aß(17-28), b)
Biotin-(G)5-Aß(12-40) and c) Biotin-(G)5-Aß(1-40).
5 10 15 20 25 30 35 40 45 50 55
0.2
0.3
0.4
0.5
0.6
Time(min)
A
bs (2
20
n m )
30.9
5 10 15 20 25 30 35 40 45 50 55
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
A
bs (2
20
n m )
Time(min)
33.6
5 10 15 20 25 30 35 40 45 50 55
0,2
0,3
0,4
0,5
0,6
0,7
Time(min)
A
bs (2
20
n m )
33.8
1000 1500 2000 2500 3000 3500 m/z
500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 m/z
1000 1500 2000 2500 3000 3500 4000 4500 m/z
A
bs (2
20
n m )
A
bs (2
20
n m )
A
bs (2
20
n m )
A
bs (2
20
n m )
Results and discussion 67
Table 6: Characteristics of antigenic peptides synthesized for ELISA
Peptide Sequence
HPLC
Rt (min)
[M+H]+
calculated found
Biotin-G5-(17-28) Biotin-GGGGGLVFFAEDVGSNK 30.9 1835.8745 1835.8717
Biotin-G5-(12-40)
Biotin-GGGGGVHHQKLVFFAEDVGSNKGAII
GLMVGGVV
33.8 3531.8352 3531.8559
Biotin-G5-(1-40)
Biotin-GGGGGDAEFRHDSGYVHHQKLVFF
AEDVGSNKGAIIGLMVGGVV
33.6 4838.3566 4838.4590
2.2.7. Characterisation of the human Aß-autoantibodies binding to amyloid
peptides by ELISA
The proteolytic excision and mass spectrometric studies employed for the
identification of the antigenic determinant recognized by the Aß-antibodies lead to
the identification of the Aß- fragments [12-40] and [21-37]. Considering these
results, a further goal of this work was to compare the antigenic binding properties
of the Aß-epitope peptides and the binding of different Aß-antibodies to N-terminal
and C-terminal ß-amyloid sequences using indirect ELISA.
In contrast to the sandwich ELISA which requires that the antigen contains 2
binding sites, one for the detection antibody and one for the antibody of interest, in
indirect ELISA the antigen is coated first to a microwell plate followed by addition
of the antigen-specific antibody and detection of the bound antibody by an HRPconjugated secondary antibody. The antigen can be immobilised directly by
adsorbtion to the microwell plate or by using a biotin coupled at the amino-terminal
end of the peptide and binding to the streptavidin- precoated plates by
noncovalent interaction. In the present work direct immobilization of the antigen to
the plate was used if comparison of the binding of different antibodies to the same
antigen was investigated. To compare the reactivity shown by an antibody to
different amyloid peptides, the biotin-streptavidin immobilization system was
employed. This approach provides the advantage of comparable extent of
immobilization at a given antigen concentration independent of peptide sequence.
Results and discussion 68
In a first experiment the binding of the Aß-antibody purified from the IgG product
(Calbiochem) to the sequences [1-40], [12-40], [1-16] and [17-28] was probed. A
pentaglycine spacer and biotin were attached to the N-terminal part of the
sequences to ensure the immobilization of the peptides on streptavidin coated
microtiter plates and suitable accessibility of the antibody to the peptides. The
peptides were added to the plates in 12 three fold serial dilutions (from 20 to
0.0001 µM) and the Aß-antibody in a constant concentration in all the wells. Aßantibody binding to the antigens was detected with an anti-human horseradish
peroxidase labeled antibody.
The plot of the OD450 vs peptide concentration is shown in the Figure 44. The Aßantibody reacted with biotin-G5-Aß(1-40) and biotin-G5-Aß(12-40) but not with
biotin-G5-Aß(1-16) and biotin-G5-Aß(17-28). These results are consistent with the
mass spectrometric data provided by the epitope identification. The binding of
biotin-G5-(12-40) was somewhat lower in comparison to biotin-G5-(1-40).
In a second experiment the binding of Aß(1-40) to the autoantibodies isolated on
the affinity column containing the complete Aß(1-40) sequence was compared with
-0,1
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
0,0001 0,0003 0,001 0,003 0,0091 0,027 0,081 0,244 0,73 2,2 6,6 20
Antigen concentration (µM)
O
D
45
0n m Biotin-GGGGG - 1DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV40
Biotin-GGGGG - 12VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
Biotin-GGGGG - 1DAEFRHDSGYEVHHQK16
Biotin-GGGGG - 17LVFFAEDVGSNKGAIIGLMVGGVV28
O
D
45
0n m Figure 44: Binding properties of Aß-autoantibody purified from the immunoglobulina gamma
preparation (Chemicon) to amyloid peptides spanning different domains of Aß(140): (○) Aß(1-16) and (X) Aß(17-28), (▲) Aß(12-40) and (■) Aß(1-40),
Background signals from wells without antigen have been substracted.
Results and discussion 69
the binding to the autoantibodies purified on the affinity column containing the
partial sequence Aß(12-40). For this experiment, Aß(1-40) was coated on the
wells at the concentration of 500 ng/well and incubated with different dilutions of
each of the autoantibodies (Figure 45). The binding of Aß(1-40) to both
autoantibodies was identical. Based on this result one can conclude that both
antibodies target the same epitope residing within the sequence Aß(12-40).
Several ELISA experiments were performed to examine binding specificity of
different samples of Aß-autoantibodies. The autoantibody samples were purified
from the immunoglobulin gamma preparation (Chemicon) and from the serum of a
healthy individual (105) and 2 Alzheimer´s Disease patients (005 and 006). In
order to investigate whether these samples display a similar preference for binding
the carboxy-terminal fragment and lack the reactivity towards the amino-terminal
end their reactivity towards biotin-G5-Aß(12-40) and biotin-G5-Aß(1-16) was
probed. For the experiment, the peptides biotin-G5-Aß(12-40) and biotin-G5-Aß(116) were applied to streptavidin coated plates using threefold serial dilutions. The
autoantibodies were incubated at a fixed concentration of 0.01 µg/µl. Bound
antibody was detected with HRP-conjugated anti-human antibody.
0
0,2
0,4
0,6
0,8
1
1,2
1:65610 21870׃ 1 7290׃ 1 2430׃ 1 810׃ 1 270׃ 1 90׃ 1 30׃ 1
Antibody dilution
O
D
45
0n m O
D
45
0n m Figure 45: Binding of Aß-antibodies purified on Cys-Aß(1-40) (▲) and Cys-Aß(12-40) (●)
affinity media to Aß(1-40) as measured by indirect ELISA. Background signals
from wells without antigen for each antibody sample and concentration have been
substracted.
Results and discussion 70
The comparison of the Aß-autoantibodies purified from the immunoglobulin
gamma preparation and from the serum of a healthy individual led to the
conclusion that there is no significant reactivity towards biotin-G5-Aß(1-16) for both
Aß-autoantibody samples. In contrast, both antibody samples showed binding to
biotin-G5-Aß(12-40) (see Figure 46).
a) b) Figure 46: Reactivity between Aß-antibody purified from (a) IgG (Chemicon) and (b) sample 105
with amyloid peptides immobilized on streptavidin coated plates (■) biotin-G5-(12-40)and
(♦) biotin-G5-(1-16). Background signals from wells without antigen have been
substracted.
1.6
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
20.06.72.20.7410.2470.0820.0270.0090.003
Antigen concentration (µM)
A
bs or ba nc e (4
50
n m )
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
20.06.72.20.7410.2470.0820.0270.0090.003
Antigen concentration (µM)
A
bs or ba nc e (4
50
n m )
Pool of healthy individuals
Healthy individual (105)
Biotin-G5-Aß(12-40)
Biotin-G5-Aß(1-16)
Biotin-G5-Aß(12-40)
Biotin-G5-Aß(1-16)
A
bs or ba nc e (4
50
n m )
A
bs or ba nc e (4
50
n m )
Results and discussion 71
The reactivity of Aß-antibodies purified from the serum of 2 AD patients (samples
005 and 006) to the peptides biotin-G5-Aß(12-40) and biotin-G5-Aß(1-16) was
compared using the same experimental setup. The result is shown in Figure 47.
Biotin-G5-Aß(12-40) reacted with both antibodies (005 and 006) in a dosedependent manner. In contrast biotin-G5-Aß(1-16), displayed no interaction to both
antibodies. These results are consistent with the epitope identification data
obtained by the epitope excision and extraction.
a) b) Figure 47: Reactivity between Aß-antibody purified from (a) sample 005 and (b) sample 006 with
amyloid peptides immobilized on streptavidin coated plates (■) biotin-G5-(12-40)and
(♦) biotin-G5-(1-16). Background signals from wells without antigen have been
substracted.
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
20.06.72.20.7410.2470.0820.0270.0090.003
Antigen concentration (µM)
-0.1
0
0.1
0.2
0.3
0.4
0.5
20.06.72.20.7410.2470.0820.0270.0090.003
Antigen Concentration (µM)
A
bs or ba nc e (4
50
n m )
A
bs or ba nc e (4
50
n m )
Alzheimer´s Disease Patient (005)
Alzheimer´s Disease Patient (006)
Biotin-G5-Aß(12-40)
Biotin-G5-Aß(1-16)
Biotin-G5-Aß(12-40)
Biotin-G5-Aß(1-16)
A
bs or ba nc e (4
50
n m )
A
bs or ba nc e (4
50
n m )
Results and discussion 72
2.2.8. Concluding discussion of the Aß-autoantibody epitope
Differences in plasma levels of Aß-antibodies between aged normal individuals
and Alzheimer´s disease patients have been reported by several authors leading
to the hypothesis that people possessing autoantibodies against Aß would be
protected against AD. In the present study the identification of the epitope
recognized by different Aß-antibodies is reported. The mass spectrometric
identification of the epitope together with the ELISA binding studies suggest that
the antibodies analysed recognize an epitope located within the amino acid
residues [21-37] and that there is no difference between the binding specificity of
the antibodies purified from healthy individuals and AD patients.
.
Considering these data it can be concluded that the autoantibodies might play a
protective role against AD, by recognition of a C-terminal epitope that inhibits the
aggregation of Aß sequences, by sequestering soluble Aß from human blood and
causing the efflux of Aß from brain to the CSF and human blood. A proposed
ß-secretase
Extracellular domain Transmembrane
domain
EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKK
γ−secretaseα-secretase
APP
1 42
ß-secretase and
γ-secretase cleavage
aggregation
1 40
1 40
140
1 40
140
Anti-(C-terminal) IgG
Figure 48: Potential mechanism of action of Aß-autoantibodies.
Results and discussion 73
mechanism for the “plaque-protecting” effect of Aß-autoantibodies is summarized
in Figure 48.
However, despite these results, it must be emphasized that the reason for the
natural occurence of Aß-antibodies in human blood is unclear at present.
Considering that autoimmunity accounts for many human diseases such as
systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, myasthenia
gravis and others it is unclear whether the Aß-reactive antibodies are autoimmune
antibodies or antibodies that display cross-reactivity to Aß. Thus, an interesting
further step in the characterization of the autoantibodies would be to test whether
the Aß-antibodies display reactivity to the compounds present in the human blood.
Results and discussion 74
2.3. Investigation of the cleavage specificity of ß-amyloid peptides by HtrA1
protease
2.3.1. Structure and biological functions of HtrA1
Many damaged proteins represent a serious hazard to the cell as they might
accumulate as large aggregates. This process has been associated with
neurodegenerative diseases, prion disease and amyloid diseases resulting from
protein misfolding and/or aggregation pathways. To prevent the inappropriate
association or aggregation of exposed hydrophobic surfaces, cells have available a
sophisticated system of molecular chaperones and proteases. These systems may
be assigned as “molecular quality control factors” facilitating protein folding and
eliminate denatured proteins that may be toxic when they accumulate in the cell [141].
The HtrA (high temperature requirement) family of proteins belongs to a highly
conserved class of serine proteases that combine the dual activities of chaperones
and proteases. Its members are classified by the combination of a catalytic domain
resembling trypsin with one or more C-terminal PDZ domains [142, 143]. PDZ
domains are protein modules responsible for substrate binding and subsequent
translocation into the catalytic cavity. Human HtrAs have been recently identified and
named HtrA1, HtrA2 and HtrA3 [144-146]. These proteases are believed to be
involved in arthritis, apoptosis, neuromuscular disorder and cancer [147]. HtrA1 is
likely to be involved in the degradation of extracellular matrix proteins which could be
important for both arthritis, tumor progression and age related diseases. Although the
cleavage site specificity of this enzyme was not yet established, there appears to be
a preference for valine and isoleucine as residue preceding the cleavage site. The
evaluation of the molecular cleavage properties of the HtrA protease toward Aßpeptides, and several transmembrane Aß-precursor peptides, using mass
spectrometric methods was the goal of this chapter.
Results and discussion 75
2.3.2. Clearance of cerebral Aß by enzymes
Clearance of cerebral Aß peptides can be achieved either by receptor-mediated
transport into the blood stream through the blood-barier, microglia, or degradation by
proteolytic enzymes [148]. Recent studies have focused on neuroglial cells as
important players in Aß metabolism. Activation of astrocytes and microglia occur
early in AD in the periphery of senile plaques [149]. Both cell types are highly
reactive to environmental changes and secrete complement proteins, inflammatory
cytokines, acute phase reactants, and proteases and their inhibitors [150, 151].
When astrocytes are plated on unfixed Aß-rich brain sections from transgenic mice
expressing human APP, Aß levels decreased with 40% within 24h [152]. Furthermore,
the exposure of astrocytes to Aß42 in cell culture supernatants led to a complete
removal and degradation of Aß within 48h. Although these findings suggest an
important role for astrocytes in Aß metabolism it is still unclear whether Aß digestion
occurs on the cell surface of astrocytes by secreted proteases or internally. Several
candidate proteases, such as insulin-degrading enzyme, neprylysin, and endothelinconverting enzyme, have been implicated in removal of Aß [148].
Immunohistochemical investigation of brains from AD patients using HtrA1 specific
antiserum revealed the presence of HtrA1 in astrocytes and cortical neurons.
Moreover, most amyloid plaques, positively stained with Congo red, were also
positive for the expression of HtrA1 [142]. In contrast, HtrA1 expression in neuroglial
components and neurons of normal adult brains was found to be very low [153]. The
presence of secreted and intracellular HtrA1 suggested that HtrA1 might have the
potential to cleave extracellular and intracellular segments of the amyloid precursor
protein.
Results and discussion 76
2.3.3. Analytical characterization of C99 and HtrA
Purified, recombinant C99 and HtrA1, used in this study, were obtained from the
group of Professor Michael Ehrmann, Cardiff University of Cardiff. Both proteins were
expressed with a C-terminal histidine-tag oligopeptide-tag for affinity-purification [142].
The molecular mass and sequence of C99 were determined by MALDI-TOF-MS, and
by high resolution ESI-FTICR-MS. The experimental values of the ions observed in
the mass spectrum matched the predicted values of the 1+ (m/z 12345.06), 2+ (m/z
6173.03) and 3+ (m/z 4115.69) ions.
Additionally, the purities of C99 and HtrA were investigated by Tris-Tricine SDSPAGE (Figure 50 a). In order to monitor the presence of low amounts of contaminant
proteins in the sample, silver staining was used. The gel revealed the presence of an
intense spot corresponding to the predicted value of the HtrA molecular mass (38371
Da) and of several contaminant proteins. This result indicated that the enzyme might
be partially degraded with time. No contaminant band was identified in the C99
sample. The absence of contaminant proteins in the mass spectrum and SDS-PAGE
of C99 provided quality assurance for the investigation of the proteolytic specificity of
HtrA1.
γ-secretase
γ-secretase

P3
APP
Extracellular domain
sAPPα
sAPPβ
ß-secretase
α-secretase
C99
C83
7701 671
71
6
71
7
1 770
1 770687
71
6
71
7
Figure 49: Proteolytic processing of
amyloid precursor protein. Cleavage
by α-secretase produces soluble
APPα and a C83-fragment which is
further cleaved by γ-secretase to
produce the non-amyloidogenic
fragment P3. Alternatively, ßsecretase cleavage of APP results in
soluble APPß and C99 which
undergoes a second cleavage by γsecretase producing ß-amyloid and a
C57-59-fragment.
Results and discussion 77
a) b)
c) 2.3.4. Mass spectrometric identification of cleavage sites in C99 and Aß
The proteolytic digestion by HtrA1 was analysed with C99 protein, in comparison to
synthetic Aß(1-40), Aß(1-42), the cytosolic C-terminal polypeptide APP(724-770) and
the N-terminal APP(661-687). In order to facilitate the comparison of the partial
sequences generated by the enzymatic digestion of the different substrates, the APP
nomenclature of the amino acid residues was used for the sequence designation of
all substrates (Table 7).
U
LM
W
(
kD )
67
45
36
29
24
20
14
26
17
14
6.5
3.5
LM
W
(
kD )
H
tr A
C
99
U
LM
W
(
kD )
LM
W
(
kD )
H
tr A
C
99
U
LM
W
(
kD )
LM
W
(
kD )
H
tr A
C
99
LM
W
(
kD )
H
tr A
C
99
[M
+H
]+
[M
+2
H
]2
+
[M
+
3H
]3
+
[M
+H
]+
[M
+2
H
]2
+
[M
+
3H
]3
+
Figure 50: Analytical characterization of the recombinant C99 through a) Tris-Tricine SDS-PAGE
and b) MALDI-TOF mass spectrometry, c) nano-ESI-FT-ICR mass spectrometry.
m/z823.9 m/z 882.6 883.1
820 840 860 880 900 m/z
[M+15H]15+
[M+14H]14+
Results and discussion 78
All proteolytic digestions were performed at a substrate/enzyme ratio of 1:50 for a
time period of 24 h. The proteolytic fragment mixtures were then desalted and
directly analysed by MALDI-FTICR-MS as described in Experimental Part. The
monoisotopic peptide masses were selected manually and used for fragment
identification by means of GPMAW program [154]. All molecular ions present in the
mass spectrum provided unequivocal identifications of the partial sequences of C99
with mass determination accuracies of <10 ppm. A specific molecular cleavage
pattern was identified for the HtrA1 digestion of C99, as shown by MALDI-MS of the
sequence of the product fragments (Figure 51). Digestion was established to occur
after residues Val-683, Gln-686, Asn-755, and Asp-672 providing degradation
products of approximately equal sequence lengths, without a particular cleavage
specificity of single amino acids.
a) To ascertain the cleavage sites, Aß- peptides C-terminal APP peptide domains were
used as additional substrates under identical digestion conditions. The same specific
[6
73
-6
83
]
[6
71
-6
83
]
[6
71
-6
86
]
[7
56
-7
70
]
[6
73
-6
83
]
[6
71
-6
83
]
[6
71
-6
86
]
[7
56
-7
70
]
MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVML
KKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNRSHHHHHH
671
770
Figure 51: Identification of htrA1 cleavage specificity in C99 by MALDI-FTICR-MS. The insert
shows the detected cleavage sites identified on the sequence of recombinant C99.
The peptide fragments identified in the proteolytic mixture are underlined.
Results and discussion 79
cleavage sites Val-683 and Gln-686 as in C99 were also found for Aß(1-40) and
Aß(1-42); however additional sites were identified in Aß(1-40) located two and three
residues further downstream. The cleavage sites at Val-683 and Asp-672 were also
identified in the APP(661-687) peptide.
a) b) [6
72
-6
83
]
[6
72
-6
86
]
[6
72
-6
88
]
[6
72
-6
89
]
[6
72
-7
11
]
[6
72
-6
83
]
[6
72
-6
86
]
[6
72
-6
88
]
[6
72
-6
89
]
[6
72
-7
11
]
711
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
672
[6
72
-6
83
]
[6
72
-6
86
]
[6
72
-7
13
]
[6
72
-6
83
]
[6
72
-6
86
]
[6
72
-7
13
]
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
672 713
Results and discussion 80
c) d) [7
35
-7
48
]
[7
24
-7
38
]
[7
56
-7
70
]
[7
49
-7
70
]
[7
24
-7
48
]
[7
39
-7
40
]
[7
35
-7
48
]
[7
24
-7
38
]
[7
56
-7
70
]
[7
49
-7
70
]
[7
24
-7
48
]
[7
39
-7
40
]
KKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN
Figure 52: Identification of htrA1 cleavage specificity in a)APP(672-711), b)APP(672-713),
c)APP(661-687) and d) APP(724-770) by MALDI-FTICR-MS. The insert shows the
detected cleavage sites identified. The peptide fragments identified in the proteolytic
mixture are underlined.
[6
61
-6
87
]
[6
67
-6
87
]
[6
73
-6
87
]
[6
70
-6
83
]
[6
75
-6
87
]
[6
70
-6
87
]
[6
61
-6
87
]
[6
67
-6
87
]
[6
73
-6
87
]
[6
70
-6
83
]
[6
75
-6
87
]
[6
70
-6
87
]
IKTEEISEVKMDAEFRHDSGYEVHHQK
661 687
Results and discussion 81
A molecular degradation pattern consistent with this cleavage selectivity was found
for the C-terminal domain of C99, by using APP(724-770) as a substrate. Although
the cleavage site at Asn-755, found in C99, was also identified in the substrate
APP(724-770), additional cleavage sites were identified at the Gly-734, Val-738 and
His-748 residues (Figure 52), indicating a shift in the substrate fitting to the protease
according to the oligo-His-Tag sequence present in the C99 polypeptide.
Taken together, these results show HtrA1 has no clear preference for P1 residues
but generates products of similar lengths ranging between 10-20 residues. The
presence of secreted and intracellular HtrA1 suggests that HtrA1 has the potential to
cleave the extracellular and intracellular segments of C99. The mass spectrometric
detected cleavage pattern is consistent with the findings that HtrA1 interferes with Aß
Table 7: Sequence assignment of the masses determined by MALDI-FT-ICR mass spectrometry
based on the correlation to tryptic fragments mass profiles of the proteins included in the
database.
Substrate Sequence [M+H]+calc. [M+H]
+
exp.
∆m in
ppm
Cleavage
site
C99 673AEFRHDSGYEV683 1309.5735 1309.5868 5 D672 V683
671MDAEFRHDSGYEV683 1555.6409 1555.6650 11 V683
671MDAEFRHDSGYEVHHQ686 1957.8173 1957.8561 16 Q686
756GYENPTYKFFEQMQN770-RSHHHHHH 2961.3062 2961.3569 15 N755
Aß(1-40) 672EFRHDSGYEV683 1424.6004 1424.6155 9 V683
672DAEFRHDSGYEV686 1826.7768 1826.8109 14 Q686
672DAEFRHDSGYEVKL688 2067.9558 2067.9782 8 L688
672DAEFRHDSGYEVKLV689 2167.0243 2167.0873 27 V689
Aß(1-42) 672EFRHDSGYEV683 1424.6004 1424.6098 2 V683
672DAEFRHDSGYEVHHQ686 1826.7768 1826.7981 9 Q686
APP(661-687) 675FRHDSGYEVHHQK687 1638.7811 1638.7897 1 E674
670KMDAEFRHDSGYEV683 1683.7359 1683.7491 4 V669 V683
673AEFRHDSGYEVHHQK687 1838.8608 1838.8733 3 D672
670KMDAEFRHDSGYEVHHQK687 2213.0232 2213.0367 3 V669
667SEVKMDAEFRHDSGYEVHHQK687 2528.0232 2528.1944 8 I666
APP(724-770) 724KKKQYTSIHHGVVEV738 1752.9682 1752.9959 12 V738
739DAAVTPEERHLSKMQQNGYENPTYKF
FEQMQN770 3829.7624 3829.7903 5 V738
735VVEVDAAVTPEERH748 1550.7736 1550.7948 9 G735 H748
724KKKQYTSIHHGVVEVDAAVTPEERH748 2858.4835 2858.5975 37 H748
749LSKMQQNGYENPTYKFFEQMQN770 2724.2472 2724.2987 16 H748
756GYENPTYKFFEQMQN770 1894.8356 1894.8734 16 N755
Results and discussion 82
production in astrocytes and the application of an HtrA1 inhibitor leads to a significant
accumulation of Aß in cell culture supernatants. HtrA1 activity could therefore reduce
formation of Aß deposits in human brains by competing with γ-secretase for
substrate and removing Aß. These results suggest that activation of protein qualitycontrol factors represents one promising general strategy for the prevention of
degenerative diseases that are based on protein aggregation and amyloid formation.
Results and discussion 83
2.4. Epitope identification of a monoclonal antibody to the H1-carbohydrate
recognition domain (H1CRD) of the asialoglycoprotein receptor
2.4.1. Structure and biological functions of H1CRD
The asialoglycoprotein receptor (ASGPR) belongs to the C-type (calcium-dependent)
class of lectins because of its requirement of calcium ions for carbohydrate binding
[155]. ASGPR occurs in the liver of all mammalian organisms that have been
examined [156, 157]. The receptor consists of a hetero-oligomer of two homologous
subunits H1 and H2 with 58 % sequence homology [158, 159]. Both subunits are
composed of a cytosolic N-terminal domain, a single transmembrane segment, a
stalk domain and a C-terminal carbohydrate recognition domain (CRD) (Figure 53).
The carbohydrate recognition domain binds galactose (Gal) and Nacetylgalactosamine (GalNAc)-terminal glycoproteins that are exposed by the
removal of a terminal sialic acid (Sia) residue [160]
a) b)
Figure 53: Schematic representation of the asialoglicoprotein receptor (ASGPR). a) a hetero-oligomer
composed of one H1 subunit and one H2 subunit; b) the four domains of the H1 subunit
cytosolic, transmembrane, the stalk and the carbohydrate recognition domain are
indicated by arrows.
The receptor provides clearance of circulating ligand glycoproteins by receptormediated endocytosis followed by degradation in the lysosome, and the receptor is
then recycled to the cell surface. While both subunits are required for the endocytosis
of ligands, the carbohydrate binding activity was shown to be associated
predominantly with H1 [161, 162]. Recent studies suggest that ASGPR is used as
entry site into hepatocytes by hepatitis B virus, Marburg virus and hepatitis A virus
[163-165]. Thus molecules that bind specifically to the carbohydrate recognition
Cytoplasmic
domain
Transmembrane
segment
Stalk
Carbohydrate recognition
domain (CRD)
Asialoglycoprotein receptor
Cell membrane
Results and discussion 84
domain such as monoclonal antibodies might exert inhibitory effects towards these
diseases by blocking the virus entry site. The epitope elucidation carried out for
antibodies that are raised against the carbohydrate recognition domain can be
expected to lead to the identification of antibodies capable of blocking the
carbohydrate recognition site within the carbohydrate recognition domain of the
subunit H1.
The carbohydrate recognition domain of subunit H1 (H1-CRD) consists of the amino
acid residues (148-291). The X-ray crystal data (available at the PDB accession
number 1dv8) describes the structure of the amino acid residues (153-280), however
the structure of the N and C-terminal residues (148-153) and (282-291) could not be
determined, possibly due to thermal disorder [166]. According to these data the H1CRD is a globular protein that comprises six ß-strands (ß1-ß6) and 2 α-helices of 10
and 11 residues respectively (Figure 54). The protein contains 7 cysteine residues for
which 2 disulfide bridges have been identified and a third disulfide bond has been
proposed. The structure contains three Ca2+ binding sites. The carbohydrate binding
site is formed by the carboxy and amino groups of the amino acid residues Gln239,
Asp241, Trp243, Glu252, Asp 253 and Asn254 [167, 168]. Although the carbohydrate
binding site resides between amino acid residues (239-253), the globular structure of
the carbohydrate recognition domain requires the complete amino acid sequence
suitable to be used as immunogen.
Figure 54: Ribbon diagram of the
H1-CRD based on the X-Ray
crystal structure (PDB accession
number 1dv8). The N and C
terminus are on the left side of the
image. The α-helices are indicated
by red arrows and the β-strands by
blue arrows. The calcium ions are
depicted by black balls. The figure
was prepared using the program
BallView 1.1.1. ß1
ß2
ß6
C
N
ß3 ß4 ß5
α2
α1
Ca2+
Results and discussion 85
2.4.2. Primary structure characterisation of H1CRD using mass spectrometric
methods
The recombinant carbohydrate recognition domain of the subunit H1 employed in the
present work was expressed in E. coli and purified by affinity chromatography using a
galactose-Sepharose affinity column, followed by size exclusion chromatography
[169]. The recombinant protein comprises the amino acid residues [148-290] of the
H1CRD and an initiator methionine residue. The amino acid residues of the
recombinant H1CRD are numbered from 1 to 145 and the same numbering format
will be used throughout par. 2.4. (Figure 55).
H1CRD(P07306) GSERTCCPVN157 WVEHERSCYW167 FSRSGKAWAD177 ADNYCRLEDA1 8 7
Recombinant
P ro te in
M1GSERTCCPVN110 WVEHERSCYW210 FSRSGKAWAD310 ADNYCRLEDA4 1 0
H1CRD(P07306 ) HLVVVTSWEE197 QKFVQHHIGP2 07 VNTWMGLHDQ217 NGPWKWVDGT227
Recombinant
Pro te in HLVVVTSWEE
510 QKFVQHHIGP6 10 VNTWMGLHDQ710 NGPWKWVDGT810
H1CRD(P07306) DYETGFKNWR237 PEQPDDWYGH247 GLGGGEDCAH257 FTDDGRWNDD267
Recombinant
Pro te in DYETGFKNWR
910 PEQPDDWYGH101 GLGGGEDCAH111 FTDDGRWNDD121
H1CRD (P07306) VCQRPYRWVC277 ETELDKASQE287 PPLL291
Recombinant
Pro te in VCQRPYRWVC
131 ETELDKASQE141 PPLL145
Figure 55: Alignment of the recombinant sequence of H1CRD to the sequence available at the SwissProt protein sequence database with the accession number P07306
The molecular mass of the intact H1CRD was initially determined by ESI-FT-ICR
mass spectrometry. For performing FTICR-MS an aliquot of the stock solution of
H1CRD was diluted with 50% methanol, 1% acetic acid in MilliQ to a final
concentration of 6 pmol/µl and the sample introduced in the electrospray source at a
flow rate of 2 µl min-1. The charge of each molecular ion present in the mass
spectrum was determined from the 1/z spacing between the isotopes as shown in the
insert on the right side of the Figure 56. The monoisotopic theoretical molecular mass
of the molecule in the oxidized state (containing 3 disulfide bridges) is 16975.5795
and in the reduced state is 16981.6263. The monoisotopic molecular mass
determined from the mass spectrum is 16921.9093, showing a mass shift of 52.5624
Results and discussion 86
to 59.7170 Da depending on the number of cysteine residues existing in reduced and
oxidized state under the conditions of the electrospray FT-ICR mass measurement.
∆m=1/11
The only modification of the sequence that would introduce a decrease of the
molecular mass approximately within the calculated mass shift interval is a missing
glycine residue (57.0215 Da). However, the expression of the protein lacking one
amino acid residue within the sequence is unlikely. A more common modification that
occurs at the expression of the proteins is the lack of the initiator methionine.
Therefore it can be hypothesised that the initiator methionine might be missing
(molecular weight decrease of 131.0405 Da) and one amino acid carries a
modification that increases the molecular mass by 72.3313-79.4781 Da. Due to the
treatment of the sample wit ß-mercaptoethanol during the extraction of the protein
from the cell lysate and during the purification steps [169] and considering the
presence of an odd number of cysteine residues within the sequence, the formation
of an adduct of H1-CRD with ß-mercaptoethanol was assumed. In Table 8 the
monoisotopic molecular mass experimentally determined is compared with the
Figure 56: ESI-FT-ICR mass spectrum of the H1CRD in 50 % methanol, 1 % acetic acid in water at a
concentration of 6 pmol/µl. The spectrum shows 8- to 13 times charged molecular ions.
The isotope-resolved mass spectrum of the 11-times charged molecular ion is shown in the
upper right panel. The insert on the left side shows the isotopic structure of the ion [M+H]+
after deconvolution of the ESI spectrum.
[M+13H]13+
[M+12H]12+
[M+11H]11+
[M+10H]10+
[M+9H]9+
[M+8H]8+
Results and discussion 87
theoretical monoisotopic masses of the protein according to the number of disulfide
bonds and the modifications discussed above.
Table 8: Comparison between the experimental determined molecular weight of the H1CRD and the
theoretical mass of the protein according to the proposed modifications
Number of
disulfide bonds
Monoisotopic molecular weight [M+H]+
H1CRD -Gly - Met - Met/+ ß-ME Measured
Reduced state 16981.6263 16924.6048 16850.5858 16926.5841
16921.9093 1 S-S bond 16979.6107 16922.5859 16848.5702 16924.5685
2 S-S bonds 16977.5951 16920.5736 16846.5546 16922.5529
3 S-S bonds 16975.5795 16918.5580 16844.5390 16920.5373
The probability that an ion contains more 13C and 15N isotopes increases with the
molecular mass therefore the intensity of the monoisotopic ions decreases. Thus the
determination of the monoisotopic mass is difficult at higher molecular masses. In
order to obtain information concerning the modification that leads to the molecular
weight decrease in the ESI-FT-ICR mass spectrum the primary structure was
investigated by proteolytic digestion of native H1CRD with trypsin followed by
MALDI-FT-ICR analysis of the tryptic fragments. The arginine and lysine residues
present in the amino acid sequence of H1-CRD are highlighted in the Figure 57a.
Twelve tryptic fragments could be formed by complete digestion of H1-CRD by
trypsin which are designated T1 to T12 (Figure 57b); the cysteine residues are also
indicated.
a) MGSERTCCPV NWVEHERSCY WFSRSGKAWA D AD N Y C R L E D 40
AHLVVVTSWE E Q K F V Q H H I G PVNTWMGLHD QNGPWKWVDG 80
TDYETGFKNW RPEQPDDWYG HGLGGGEDCA HFTDDGRWND 120
DVCQRPYRWV CETELDK ASQ EPPLL
b) Figure 57: (a) Amino acid sequence of the carbohydrate recognition domain of the
asialoglycoprotein receptor subunit H1CRD; b) Schematic representation of the
trypsin cleavage sites, the tryptic peptide fragments and the location of the cysteine
residues.
1 5 17 24 28 37 53 76 88 117 128 137 145
C7C8 C19 C36 C109 C123 C131
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
Results and discussion 88
The protein was digested for 5h at 37° using an enzyme to substrate ratio of 1:50.
The mass spectra of the tryptic digest are shown in Figure 58 and the fragments
identified are summarized in Table 9.
Table 9: Molecular mass and amino acid sequence of the peptides obtained in the MALDI-FT-ICR
mass spectrum by tryptic digestion of the native H1CRD
[M+H]+exp [M+H]
+
exp ∆m in
ppm
Tryptic fragment Sequence
948.409 948.4033 6.0 T3 18SCYWFSR24
1122.511 1122.5136 2.3 T11 129WVCETELDK137
1184.480 1184.4789 0.9 T5 28AWADADNYCR37
1417.619 1417.6270 6 T8 77WVDGTDYETGFK88
1451.679 1451.6485 21 T10 118WNDDVCQRPYR128
1470.615 1470.6253 7.0 T2 disulfide bridge 6TCCPVNWVEHER17
1883.001 1882.9545 24.6 T5+T6 28AWADADNYCRLEDAHLVVVTSWEEQK53
2303.980 2303.9694 4.6 T5/T11
disulfide bridge
28AWADADNYCR37
129WVCETELDK
2698.298 2698.3096 4.3 T7 54FVQHHIGPVNTWMGLHDQNGPWK76
3287.319 3287.3620 13.0 T9 89NWRPEQPDDWYGHGLGGEDCAHFTDDGR117
The MALDI-FT-ICR mass spectra of the peptide mixture resulted after tryptic
digestion of native H1CRD contained the tryptic fragments T3, T5, T7, T8, T9, T10
and T11. The fragments T5 and T11 are linked by a disulfide bridge and thus
established between Cys36 and Cys131. The monoisotopic molecular mass ([M+H]+
=2303.980) of the two tryptic fragments T5 and T11 bound through the disulfide
Figure 58: MALDI-FT-ICR mass spectra of the peptide mixture obtained by tryptic digestion of the
native H1CRD. The fragments containing a disulfide bridge are indicated in red. The
spectra were recorded by adjusting the hexapole parameters to get optimum
sensitivity for the mass range of interest: (a) time of flight delay 2500 us, RF amplitude
2.5 and (b) time of flight delay 3500 and RF amplitude 3.5.
T3
T11
T5
T8 T5+T6
T5+T11
T2
T5
T8
T2
T5+T6
T5+T11
T7
T9
Results and discussion 89
bridge was determined with an accuracy of 4.6 ppm. However, the molecular ions of
the free peptide fragments T5 and T11 were also identified in the mass spectrum
indicating that the disulfide bridge is not stable under the conditions of sample
preparation and measurement. Furthermore, the measured mass of T2
([M+H]+=1470.619) was 2.02 Da lower than the theoretical mass
([M+H]+=1472.6409). The fragment T2 contains two vicinal cysteine residues Cys7
and Cys8, thus showing a disulfide bridge accounting for the 2.02 Da difference.
To confirm the identity of the fragment T2 and the presence of the vicinal disulfide
bridge the cysteine residues were reduced with DTT and alkylated with
iodoacetamide as described in the Experimental Section. The alkylated H1CRD was
subjected to proteolytic digestion with trypsin and the peptide fragments resulted
were analysed by MALDI-FT-ICR (Figure 59). The peptides were identified with an
average mass error of 4.5 ppm. The fragments T3, T11 and T10 containing one
alkylated cysteine residue and the fragment T2 containing 2 alkylated cysteine
residues were found at the expected molecular masses (Table 10), thus confirming
the vicinal disulfide bridge in the native H1CRD.
T 8
T 2 * *
T 3 *
T 1 1
T 1 0 *
T 1 1 * + T 1 2
Figure 59: MALDI-FT-ICR mass spectrum of the peptide mixture obtained by tryptic digestion of
the alkylated H1CRD. The asterisk indicates a carbamidomethyl modification at
cysteine.
Results and discussion 90
Table 10: Molecular weights and amino acid sequence of the peptides obtained in the MALDI-FT-ICR
mass spectrum by tryptic digestion of the native H1CRD
[M+H]+exp [M+H]
+
calc. ∆m in
ppm
Tryptic fragment Sequence
1005.415 1005.4245 9.4 T3 18SCYWFSR24
1179.529 1179.5351 5.1 T11 129WVCETELDK137
1417.618 1417.6270 6.3 T8 77WVDGTDYETGFK88
1508.671 1508.6699 0.7 T10 118WNDDVCQRPYR128
1586.681 1586.6839 1.8 T2 6TCCPVNWVEHER17
1882.962 1882.9545 3.9 T6 LEDAHLVVVTSWEEQK
2014.988 2014.9790 4.4 T11+T12 129WVCETELDKASQEPPLL145
The identification of the disulfide bond Cys36-Cys131 is in agreement with the X-ray
crystal data [166]. A a second linkage described to occur between Cys109 and
Cys123 was not detectable under the conditions employed and the tryptic fragments
T9 containing the residue Cys109 and T10 containing the residue Cys123 were
identified separately in the mass spectrum of the tryptic digest from the native protein.
The third disulfide bridge identified between Cys7 and Cys 8 is not consistent with
previous data which are based on the X-ray crystal structure describing a disulfide
bridge between Cys8 and Cys19. A possible explanation for the vicinal disulfide
bridge identified in the mass spectra of the tryptic digest may be a rearrangement of
the disulfide bonds after the reduction followed by renaturation of the H1CRD during
the purification steps.
The formation of a disulfide bond between adjacent cysteine residues is a relatively
rare structural element which is usually accompanied by the formation of a ß-type
turn of the protein backbone. In the last few years, vicinal disulfides have been
identified and structurally characterized in a variety of proteins including enzymes,
receptors and toxins [170-178]. Recently, non-native vicinal disulfide bonds were
observed during the oxidative folding of the 32-residue Amaranthus α-amylase
inhibitor [174], and during the synthesis of α-conotoxin [179]. Using the data on XxxCys-Cys-Yyy amino acid sequence of the proteins that contain a vicinal disulfide
bridge taken from Brookhaven Protein Data Bank, Hudaky and coworkers [180]
showed that Ser, Thr, Leu, Gly, Glu and Pro, Asp, Asn, Arg are the most frequent
amino acid residues in positions Xxx and Yyy respectively. Noteworthy, in the case of
the H1CRD, Thr and Pro are present in the positions Xxx and Yyy (Thr-Cys-Cys-Pro).
Results and discussion 91
The structural data obtained from the tryptic digestion of both native and denatured
form resulting by reduction and alkylation of the cysteine residues are summarized in
the Figure 60.
Figure 60: Schematic representation of the structural information obtained by tryptic digest of the H1CRD and mass spectrometric determination of the resulting peptide fragments. The tryptic
fragments that were identified in the mass spectra are highlighted in grey. The 2 disulfide
bonds that were identified are indicated by a connector line between the cysteine residues
involved in the formation of the bridge.
The peptides identified in the tryptic digest of the native and alkylated H1CRD cover
approximately 95 % of the total sequence. The ions corresponding to the short
peptide fragments T1 and T4 were not found in the mass spectra, suggesting that the
molecular mass difference observed in the ESI-FT-ICR mass spectrum of the intact
native H1CRD might be due to modifications within this domain. The trypsin cleavage
at the amino acid 5 confirms that the fragment T1 ends at Arg or Lys. This fragment
should contain 5 amino acid residues and could not be observed in the mass spectra
of the tryptic mixture. In order to obtain longer amino acid sequences, the reduced
and alkylated H1CRD was digested with LysC. The cleavage sites of LysC are
indicated in the Figure 61 and the digestion fragments are designated L1 to L6.
The Figure 62 (a) shows the base peak chromatogram obtained from the separation
of 5 µg H1CRD fragment mixture produced from the digestion with LysC. Separation
was performed using an Agilent 1100 HPLC with the mobile phase consisting of 0.1%
formic acid: 0.1% formic acid in ACN. Gradient separation was used on a 150 mm x
4.6mm x 3µm Discovery RP-18 column at a flow rate of 50 µl/min.
Figure 61: Amino acid sequence of the carbohydrate recognition domain of the asialoglycoprotein
receptor subunit H1 (A); Schematic representation of the LysC cleavage sites and the
tryptic peptide fragments (B).
1 5 17 24 28 37 53 76 88 117 128 137 145
C7 C8
C19
C36
C109 C123
C131
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
1 37 53 76 88 137 145
C7C8 C19 C36 C109 C123 C131
L1 L2 L3 L4 L5 L6
Results and discussion 92
Table 11: Summary of LC-MS data for LysC digested H1CRD. The mass spectra are shown in the
figures 61 and 62
LC
Rt(min)
Measured
m/z
Calculated
m/z
∆m
(Da)
Charge Fragment Amino acid sequence
33.5 709.298 709.317 0.02 2+ L4 WVDGTDYETGFK
34.4 854.424 854.461 0.04 1+ L6 ASQEPPLL
427.719 427.734 0.02 2+ L6 ASQEPPLL
34.7 679.724 679.331 0.14 4+ L3a FVQHHIGPVNTWMGLHDQNGPWK
543.933 543.666 0.27 5+ L3a FVQHHIGPVNTWMGLHDQNGPWK
35.6 819.512 819.364 0.15 4+ L1b GSERTCCPVNWVHERSCYWFSRSGK
655.946 655.693 0.25 5+ L1b GSERTCCPVNWVHERSCYWFSRSGK
546.654 546.578 0.07 6+ L1b GSERTCCPVNWVHERSCYWFSRSGK
36.3 675.559 675.332 0.23 4+ L3 FVQHHIGPVNTWMGLHDQNGPWK
540.718 540.467 0.25 5+ L3 FVQHHIGPVNTWMGLHDQNGPWK
38.6 1036.413 1035.817 0.6 3+ L2 AWADADNYCRLEDAHLVVVTSWEEQK
777.423 777.114 0.3 4+ L2 AWADADNYCRLEDAHLVVVTSWEEQK
622.139 621.893 0.25 5+ L2 AWADADNYCRLEDAHLVVVTSWEEQK
a Methionine oxidized
b N-terminal methionine missing
Results and discussion 93
a) b) c) 54
6.
65
4
65
5.
94
6
81
9.
51
2
92
4.
52
9
11
0
0.
2
62
65
5.
94
6
200 400 600 800 1000 1200 m/z
[M+6H]6+
[M+5H]5+
[M+4H]5+54
6.
65
4
65
5.
94
6
81
9.
51
2
92
4.
52
9
11
0
0.
2
62
65
5.
94
6
Figure 62: (a) Base peak chromatogram obtained from LC-MS separation of the peptide mixture
produced by proteolytic digestion of H1CRD using LysC; (b) ESI-IT mass spectrum of the
peptide characterized by a retention time of 35.6 min. The multiple charged ions
correspond to the L1 peptide fragment lacking methionine; (c) MS/MS spectrum of the
[M+5H]5+ ion of the peptide eluted after 35.6 min. Fragmentation of the amide bond provides y8,
y10, y11 fragment ions which demonstrate the correct identification of the L1 fragment
missing the Met-1. residue.
G S E R T C C P V N W V H E R S C Y W F S R S G K
y11y10y8
24
2.
12
5
25
8.
85
9
30
1.
19
9
32
5.
22
2
34
5.
68
3
36
3.
15
0
39
1.
03
2
42
6.
37
0
47
1.
52
7
48
7.
19
8
51
1.
57
5 53
4.
11
1
55
8.
69
2
59
9.
38
6
61
0.
85
1
62
5.
65
6
64
5.
82
8
66
5.
88
7
69
3.
20
3 70
5.
83
0
71
8.
21
4
74
3.
21
1
76
5.
83
2
78
3.
98
1
80
8.
82
4
85
1.
18
0 86
2.
26
9
88
7.
77
8
90
0.
17
2
91
9.
45
9
93
9.
25
6
96
4.
72
2
99
0.
25
6
10
50
.3
37
10
62
.8
29
11
14
.8
26
11
32
.3
16
11
51
.2
59
11
70
.8
43
200 300 400 500 600 700 800 900 1000 1100 m/z
y10
y11
y8 70
5.
24
2
70
5.
57
5
70
5.
83
0
705.0 706.0 m/z
74
3.
21
1
74
3.
61
5
74
3.
93
7
74
4.
19
8
743.0 744.0
m/z
24
2.
12
5
25
8.
85
9
30
1.
19
9
32
5.
22
2
34
5.
68
3
36
3.
15
0
39
1.
03
2
42
6.
37
0
47
1.
52
7
48
7.
19
8
51
1.
57
5 53
4.
11
1
55
8.
69
2
59
9.
38
6
61
0.
85
1
62
5.
65
6
64
5.
82
8
66
5.
88
7
69
3.
20
3 70
5.
83
0
71
8.
21
4
74
3.
21
1
76
5.
83
2
78
3.
98
1
80
8.
82
4
85
1.
18
0 86
2.
26
9
88
7.
77
8
90
0.
17
2
91
9.
45
9
93
9.
25
6
96
4.
72
2
99
0.
25
6
10
50
.3
37
10
62
.8
29
11
14
.8
26
11
32
.3
16
11
51
.2
59
11
70
.8
43
70
5.
24
2
70
5.
57
5
70
5.
83
0
70
5.
24
2
70
5.
57
5
70
5.
83
0
74
3.
21
1
74
3.
61
5
74
3.
93
7
74
4.
19
8
74
3.
21
1
74
3.
61
5
74
3.
93
7
74
4.
19
8
5 10 15 20 25 30 35 40 45 50 Time [min]
0
1
2
3
7x10
Intens.
33.5
38.6
36.3
35.6
34.4
34.7
Results and discussion 94
Figure 63: LC-ESI-IT mass spectra of the peptide fragments eluted at the retention times 38.6 min
(a), 36.3 min (b), 33.5 min (c) and 34.4 min (e); LC-ESI-IT MS/MS spectrum of the
precursor ion [M+2H]2+ = 709.298 (d); LC-ESI-IT MS/MS spectrum of the precursor ion
[M+2H]2+ = 427.719 (f).
709.298
720.277
731.270
400 500 600 700 800 900 1000m/z
[M+2H]2+
[M+H+Na]2+
[M+2Na]2+
WVDGTDYETGFK
427.719
438.728
449.727
854.424
876.413
898.401
920.348
300 400 500 600 700 800 900 1000 1100 1200m/z
[M+2H]2+
[M+H]+
[M+Na]+
[M+2Na-H]+
[M+Na+H]2+
[M+2Na]2+
ASQEPPLL
y6 b4 b4-18
y5 b5 b7 A S Q E P P L L
y6y5
b4b5 b7
342.107
398.006
416.114
439.229
513.040 611.259 687.878
723.287
300 350 400 450 500 550 600 650 700 m/z
540.718
675.559
400 500 600 700 800 900 1000 m/z
[M+5H]5+
[M+4H]4+
FVQHHIGPVNTWMGLHDQNGPWKb)
622.139
777.423
1036.413
500 600 700 800 900 100
0
1100m/z
[M+4H]4+
[M+3H]3+[M+5H]5+
AWADADNYCRLEDAHLVVVTSWEEQKa)
c) 241.098
200
258.117
452.162
744.230
859.259 1017.248
1114.325
1132.271
1213.260
1232.375
300 400 500 600 700 800 900 1000 1100 1200 m/z
y9 y7 y6 y4
y3 y3-18
y2 y2-18
452.162
453.230
452.0 453.0 m/z
W V D G T D Y E T G F K
y9y7y6y4y3y2
d) e) f)
Results and discussion 95
The mass spectra provided unambiguous identification of the fragments L2, L3, L4
and L6 (Table 11). The identification of the peptide characterised by a retention time
of 35.6 min was based on the 4-6-fold charged ions as shown in the Figure 62 B. The
measured mass of this peptide matched the calculated mass of the fragment L1
lacking the N-terminal mehionine. The MS/MS spectrum of the 5-fold charged ion
confirmed this identification. Removal of the initiator methionine by
methionylaminopeptidase was observed to occur if the side chain of the following
amino acid is small as is the case for Gly, Ala, Thr, Pro, Ser and Val [181]. Cleavage
probability was found the highest for Gly (97%) as is the case of H1CRD.
The lack of the N-terminal methionine decreases the expected monoisotopic mass of
the intact H1CRD in the reduced state from 16981.6263 to 16850.5858. The mass
identified in the electrospray mass spectrum of the intact H1CRD is 16921.9093.
Based on these data the intact H1CRD lacking the N-terminal methionine measured
in the electrospray spectrum contains a modification which accounts for the
difference of approximately 72-79 Da which, however was not observed in the
detailed analysis of the proteolytic digestion mixtures produced by trypsin and LysC.
However, although the structure of the peaks indicate that the sample is
homogeneous, a precise determination of the monoisotopic mass is not possible as
previously explained. Moreover, considering that the preferential sites of protonation
are the N-terminus of the protein, and the amino acids Lys, Arg and His, there are 21
protonation sites within the sequence and the higher charge state observed in the
electrospray mass spectrum is 13. Therefore we can assume that the protein is
partially denatured and the number of disulfide bridges cannot be unambiguously
identified. Due to the treatment with ß-mercaptoethanol during the purification steps a
plausible explanation for this result is the formation of an adduct with ßmercaptoethanol which could introduce a modification of 76 Da at the seventh
cysteine residue and which has been previously described to occur [182].
Results and discussion 96
2.4.3. Epitope identification of a monoclonal antibody to H1CRD
Following the structural characterization of the H1-CRD, the epitope excision and
extraction methods were applied in order to determine the structure of the epitope
recognized by a monoclonal antibody clone B01.4 raised against H1CRD. The
epitope identification experiments were carried out (i) with the native antigen protein
and (ii) with the denatured protein obtained by reduction and alkylation of the
cysteine residues.
An affinity column was prepared by immobilising 300 µg of the monoclonal antibody
clone B01.4 on Sepharose according to the procedure described in the experimental
part. Epitope excision mass spectrometry was carried out with 50 µg H1CRD
(antibody:antigen molar ratio 1:1) solubilised in washing buffer containing 50 mM Tris,
20 mM CaCl2, pH 8.0 . After 2h incubation, the excess of antigen was removed with
10 ml of washing buffer followed by addition of trypsin (enzyme to substrate ratio
1:50) and incubation for 5 hours at 37°C. The digestion of the protein in solution
using identical experimental conditions has been described in the previous section
2.4.2. The supernatant containing non-bound tryptic peptides was collected and the
column was washed with 30 ml buffer. Affinity bound peptides were eluted with 0.1%
TFA. The high resolution mass spectrum aquired for the affinity bound peptides
provided the identification of the tryptic fragments T2; with the sequence
TCCPVNWVEHER (containing the vicinal disulfide bridge) at m/z 1470.6300 and the
tryptic peptide T3 with the sequence SCYWFSR at m/z 948.4119. The two
sequences identified are located contiguous at the N-terminal domain of the H1-CRD
as illustrated in the Figure 64.
Results and discussion 97
To investigate whether the cysteine residues present within the sequence of H1CRD
play a role in the interaction with the antibody, the epitope excision was performed
with the alkylated antigen (see Figure 65). The molecular mass determination of the
peptide fragments present in the elution fraction revealed the presence of the
identical tryptic fragments T2 m/z 1586.6586, T3 m/z 1005.4214, T2+T3 m/z
2573.0010 and additionally T4+T5 m/z 1513.6260.
Epitope extraction was performed by applying to the affinity media 50 µg peptide
mixture resulting from the digestion of the alkylated H1CRD with trypsin. After 2h
incubation the non-bound peptide fragments were removed and the column was
washed with 30 ml washing buffer. The epitope containing peptides were eluted and
the sample was analysed by MALDI-FT-ICR mass spectrometry. Consistent with the
previous result the masses of the most abundant ions identified in the mass spectrum
of the elution fraction correspond to the N-terminal fragments T2 and T3 (Figure 66).
T2
T3
Figure 64: MALDI-FT-ICR mass spectrum of the H1CRD epitope containing tryptic peptides after
epitope excision with native H1CRD. The insert shows the sequence of H1-CRD. The
epitope peptides are underlined.
1 5 17 24 28 37 53 76 88 117 128 137 145
C7 C8
C19
C36
C109 C123
C131
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
Results and discussion 98
Figure 65: MALDI-FT-ICR mass spectrum of the H1CRD epitope containing tryptic peptides after
epitope excision with alkylated H1CRD.
Figure 66: MALDI-FT-ICR mass spectrum of the H1CRD epitope containing tryptic peptides
after epitope extraction with alkylated H1CRD. The most intense ions correspond to
the tryptic fragments T2 and T3. The asterisk indicates a carbamidomethyl
modification at cysteine.
1005.4214
1513.6260
1586.6586
2573.0010
T2
T3
T2+T3
T4+T5
T2**
T3*
T3
T2*
T4+ T5
T5
1 5 17 24 28 37 53 76 88 117 128 137 145
C7 C8 C19 C36 C109 C123 C131
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
Results and discussion 99
Interestingly, two tryptic fragments located contiguously within the sequence of
H1CRD are detected in the mass spectra of the elution fraction. This result indicates
that the Arg-17 is not involved in the interaction with the antibody. In the Figure 67 it
can be observed that all the amino acid residues of the fragment T2 are accessible at
the surface of the protein. The first 2 amino acids Tre-6 and Cys-7 are not included in
the X-ray structure. The location of Arg-17 together with Glu-16 within the loop
connecting the ß1-strand (13VGH15) and ß2-strand (18SCYWF24) favours probably the
cleavage by trypsin. The first 3 residues included in the fragment T3, Ser-18, Cys-19
and Tyr-20 following the Arg-17 are not surface accessible while the last 4 amino
acids Trp-21, Phe-22, Ser-23 and Arg-24 are exposed.
Figure 67:. Structure model of H1CRD indicating the ribbon diagram and the accessible surface area
of the tryptic fragment T3 (a) and T2 (b) identified by MS as epitope containing peptides.
The model structures were prepared with BallView v1.1.1. based on the X-Ray crystal
structure of the H1CRD (PDB entry 1DV8).The location of the first 2 amino acids of the T2
was not described in the X-Ray crystal structure.
T3
Arg24
Phe22
Ser23
Trp21
b) SCYWFSR
T2
Cys8Val10
Pro9 Arg17
Trp12Asn11
Glu14
Val13
His15
Glu16
a) (TC)CPVNWVEHER
Results and discussion 100
The borderline between continuous and discontinuous epitopes is not clearly defined
if we consider that the continuous epitopes might contain residues that are not
participating at the interaction and discontinuous epitopes contain several continuous
stretches of contiguous amino acids brought together by the folding of the protein
chain [13]. However experimental data showing linear peptides harbouring a
sufficient number of residues of the discontinuous epitope to enable the binding are
rare.
2.4.4. Affinity of synthetic epitope peptides to the monoclonal antibody
Based on the identification of the 2 epitope containing peptides by mass
spectrometry and the information provided by the structural model a further aim of
the study was the investigation of the binding of each peptide to the antibody.
The peptides T2, T3 and T2+T3 were synthesised by solid phase peptide synthesis
according to the Fmoc protocol as described in the experimental part. In order to
avoid the formation of a disulfide bridge after the deprotection and during purification,
the peptides were synthesized using cysteine residues having the functional group
permanently protected by alkylation with acetamidomethyl (Acm) (Table 12).
Table 12: Summary of the peptides syntesised by SPPS
Peptide
No Tryptic
fragment
Sequence [M+H]+exp. [M+H]
+
calc.
1 T2** TC*C*PVNWVEHER 1613.72 1613.84
2 T3* SC*YWFSR 1017.49 1017.49
3 T2+T3*** TC*C*PVNWVEHERSC*YWFSR 2614.13 2614.27
*The asterisk indicates an acetamidomethyl protecting group at cysteine
The interaction between the peptides synthesized and the B01.4 monoclonal
antibody was investigated by affinity-mass spectrometry. In a first set of experiments
the peptides were dissolved in equimolar amounts in 50 mM Tris, 20 mM CaCl2, pH 8
and the mixture was exposed to the antibody affinity media. After one hour of
incubation at room temperature the column was washed with 30 ml buffer and the
complex was dissociated under acidic conditions. The results of the experiments are
presented in the Figure 68.
Results and discussion 101
a) b)
MALDI-FT-ICR mass spectra of the elution fraction provided the identification of ions
corresponding to both peptides T2** and T3*, [M+H]+ 1613.7284 and [M+H]+
1018.4534 respectively. An additional ion [M+H]+ 1542.6918 corresponding to the
molecular weight of the peptide T2** lacking one acetamidomethyl cysteine
protecting group was identified in the spectrum (Figure 68 a).
An alternative experiment was performed by applying to the affinity media the longer
peptide containing both T2 and T3 sequences with the cysteines protected by
acetamidomethyl groups. The ion corresponding to the intact peptide [M+H]+
2614.1926 was identified in the elution fraction with lower intensity due to the
presence of the ions formed by removal of one [M+H]+ 2543.1198, two [M+H]+
2472.0962 and three [M+H]+ 2401.0860 acetamidomethyl groups (Figure 68 b).
In a third set of experiments each peptide was allowed to interact individually with the
antibody affinity media. The peptide was dissolved in 50 mM Tris, 20 mM CaCl2, pH 8
at a final concentration of 0.2 µg/µl and 10 µg of the peptide were added to the
column.
Figure 68: MALDI-FT-ICR mass spectra of the elution fractions of the synthetic peptides T3* and
T2** applied in equimolar mixture to the affinity column (a) and of the longer amino
acid sequence containing both the T2** and T3* fragments.
T2**
T3*
T2+T3***
T2+T3**
T2+T3*
T2+T3
T2*
Results and discussion 102
a) b)
The MALDI-FT-ICR mass spectra of the elution fractions contain the peptides T2**
and T3* respectively (Figure 69). These results suggest that each of the peptides is
able to bind to the antibody in the absence of the other epitope containing peptide.
Together with the epitope excision and extraction information, and the structural data
provided by the X-ray crystal structure these results support the identification of the
peptide sequence [6-24] (TCCPVNWVEHERSCYWFSR) as a conformational
epitope. However due to the ability of each of the peptides [6-17] and [18-24] to bind
individually to the antibody, each of these peptides could be considered a continuous
epitope containing peptide.
In conclusion the investigation of the epitope recognized by the monoclonal antibody
B01.4 indicates that the recognition structure is located in the opposite side of the
molecule in regard to the carbohydrate recognition site (Arg-236 to Cys-268).
Therefore, this antibody is expected to be ineffective in blocking the binding of the
carbohydrate.
Figure 69: MALDI-FT-ICR mass spectra of the elution fractions of the synthetic peptides T3* (a)
and T2** (b).
T2**T3*
T2*
Experimental part 103
3. EXPERIMENTAL PART
3.1. Proteins, Enzymes and Antibodies
Human angiotensin II, human bradykinin, human angiotensin I, human neurotensin,
human adrenocorticotropic hormone (ACTH), bovine insulin ß-chain oxidized, bovine
insulin, myoglobin, lyzozym, bovine serum albumin: Sigma.
Anti-Aß(1-17) monoclonal antibody clone 6E10: Chemicon; horseradish peroxidase
(HRP) goat anti-mouse IgG, HRP-anti-biotin antibody, HRP goat anti-human IgG:
Jackson Immunoresearch. Alkaline phosphatase-conjugated goat anti-mouse
antibody, monoclonal anti-Aß(1-12) clone Bam 10 (ascite fluid): Sigma
Sequence grade TPCK-modified trypsin, Porcine: Promega. Pronase, Streptomyces
griseus: Calbiochem, α-Chymotrypsin TLCK-treated, Endoproteinase Glu-C, strain
V8: Sigma, Endoproteinase Lys-C, sequencing grade: Roche
3.2. Materials and reagents
The following commercially available reagents were used in this work: N-α-Fmoc
protected amino acids, NovaSyn TGR resin: NovaBiochem or Reanal. D-(+)-Biotin,
CHAPS: Calbiochem; t-buthylmethylether,N-methylmorpholine (NMM), piperidine,
1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), trifluoroacetic acid (TFA), triethylsilane,
triisopropylsilane, iodoacetamide, agarose, bromophenol Blue, TEMED: Fluka, N,Ndimethylformamide (DMF): Acros Organics; Hydrochloric acid, sodium hydroxide,
urea, thiourea: Merck, deionized water: Millipore, acetic anhydride, disodium
hydrogen phosphate-2-hydrate (Na2HPO4): Riedel-de Haen; ethanol, acetonitrile
(MeCN), tris-(hydroxymethyl)-aminomethane (Tris): Roth; activated CH-Sepharose
4B, polyoxyethylensorbitanmonolaureat (Tween20), 2,2,2-trifluoroethanol (TFE), αcyano-4-hydroxycinnamic acid (HCCA), diethanolamine, dimethylsulfoxide (DMSO),
Coomassie Brilliant Blue G250: Sigma; Immobilised pH gradient (IPG) strips 3-10
and 6-11: BioRad and Sigma respectively. Carrier ampholytes (Servalyt 3-10):
Serva. 1,4-dithio-DL-treitol (DTT), acrylamide/bis solution (29.22% acrylamide, 0.78%
N,N´-methylenebisacrylamide): Genaxxon.
Experimental part 104
3.3. Solid phase peptide synthesis
The peptide sequences described in this work were synthesized by SPPS according
to Fmoc/tBu strategy using a semiautomated peptide synthesizer EPS 221 (Abimed,
Germany). The peptide synthesizer is based on a pipetting robot operated from a
keypad controller with a disc drive. Separate software allows the modification of the
program on a standard PC according to the scale of the synthesis and the sequencerelated difficulties of the peptide to be synthesized.
The syntheses of all peptides used in this work were performed on a NovaSyn TGR
resin which consists of hydroxyethyl-polystyrene beads onto which polyethylenglycol
(PEG) chains have been grafted. The resin is functionalized with the acide-labile Rink
amide linker providing amidated carboxy-terminal peptides after cleavage (Figure 70).
The level of resin substitution is usually about 200 micromoles per gram which
translates into amounts of 125, 250 and 500 mg resin per column for the synthesis
scales of 25, 50 and 100 micromoles respectively. Before the beginning of the
synthesis the resin is washed with 50 ml of DMF followed by 15 minutes incubation.
This step ensures a good solvation and accessibility of the growing peptide chains.
The amino acids used were N(α)-fluorenylmethyloxycarbonyl (Fmoc) protected. The
side-chain protecting groups are described in the appendix 2. For the synthesis, the
amino acids were weighted considering a fivefold molar excess relative to the
amount of resin.
Activation: The amino acid to be added is activated by addition of an equimolar
amount of activator and two equivalents of catalytic base. Both reagents are used as
stock solutions with a concentration of:
• 0.9 mmol/ml PyBOP
O
O
N
H
NH2OCH3
H3CO
PEG P
Figure 70: Structure of NovaSyn TGR resin.
Experimental part 105
• 4 mmol/ml for N-methyl morpholine base (NMM)
Deprotection of the N(α)-amino group: Fmoc group removal of the last amino acid
coupled to the peptide chain is achieved by addition of 2% 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU). 2% piperidine is also added when using DBU in order to
scavenge the dibenzofulvene produced on Fmoc removal, and to prevent alkylation
of resin amino groups.
NR 3̀ `
HNR 3̀ `
N
N
N
O P(NR 2`)3
N
C
H
R
O
O
N
N
N
H
O P(NR 2̀ )3
N
C
H
R2
O
OH
H
Fmoc
N
C
H
R
O
O
H
Fmoc
2
N C
H
R2
O
O
H
P(NR 2̀ )3Fmoc
Fmoc
2
N
N
N
CH 2
NH 2 C
H
R1
O
Linker
C
H 2
H
O
O
N
H
C
H
R1
O
Linker
N
C
H
R2
O
N
H
C
H
R1
O
H
Fmoc
Linker
Figure 71: Schematic representation of solid phase peptide synthesis according to the Fmoc
approach.
Experimental part 106
The coupling reaction: The solubilized and activated amino acid is added to the
resin and incubated for 30 to 50 min. Depending on the amino acid sequence, a
double coupling procedure might be employed by solubilisation of a second cartridge
containing the same amino acid followed by addition to the resin and incubation.
Biotinylation of peptides on-resin appears to be an attractive method for preparing
peptides with biotinyl moiety at the amino-terminus. Biotinylation is performed on the
peptide synthesizer using a five fold molar excess of (+) -Biotin to the amount of resin.
The deprotection of the Fmoc group carried by the last amino acid coupled as well as
the activation and coupling of the biotin are achieved using the same solutions and
reaction time used at the elongation of the peptide chain.
Cleavage of the peptides from the resin: The peptides were cleaved from the resin
using a cleavage solution containing 95% TFA as cleavage reagent and 2.5%
triethylsilan and 2.5% deionized water as scavengers for 2-3 hours at room
temperature. After cleavage, the solution containing resin and free peptide was
filtrated to remove the resin and washed twice with 1 ml TFA. The peptide present in
the filtrate was precipitated using 10 volumes of cold tert-butyl-methyl-ether over the
volume of filtrate. The precipitate was filtrate, then the solid material was washed
three times with diethylether (10 ml) and dissolved in 5% acetic acid (aqueous
solution) prior to freeze-drying.
3.4. Chromatographic and electrophoretic separation methods
3.4.1. Reversed phase-high performance liquid chromatography
Reversed phase chromatography is one of the most powerful separation techniques
for peptides and proteins. Polypeptides which differ by a single amino acid residue
NN
S
O
HH
(CH2)4
O
OH H2N+ Peptide Linker
NN
S
O
HH
(CH2)4
O
NH Peptide Linker
Figure 72: Schematic representation of peptide biotinylation on resin.
Experimental part 107
can often be separated by RP-HPLC. The sample is introduced into the HPLC
column via a manual injector. The silanol groups of the support are chemically
derivatized with hydrophobic alkyl chains (C4, C8, C18) that interact with the
hydrophobic moieties of the analyte.
Polypeptides are eluted from the reverse phase column with aqueous solvents
containing an ionic modifier to adjust the pH and an organic modifier to displace and
elute the peptide. An increasing gradient in the organic modifier is applied by using a
two-phase mobile system:
Solvent A: 0.1 % (v/v) TFA in MilliQ
Solvent B: 0.1% (v/v) TFA, 80% (v/v) acetonitrile in MilliQ
The solutions were thouroughly deaerated prior to use by vaccum combined with
sonication. The sample was dissolved in solvents that are compatible with the mobile
phase (usually the components of the gradient starting point) to avoid precipitation in
the pores of the column packing. To prevent column damage the sample was
centrifuged before injection.
Analytical RP-HPLC was performed on a Bio-Rad system (Bio-Rad Laboratories,
Richmond CA) using different columns depending on the hydrophobicity of the
sample to be analysed: Vydac C4 column (250×4.6 mm I.D.) with 5 µm silica (300 Å
pore size) (Hesperia CA); analytical Nucleosil 300-7 C18 column (250×4 mm I.D.) with
7 µm silica and 300 Å pore size (Macherey-Nagel, Düren, Germany).
Semipreparative and preparative RP-HPLC were carried out on a Knauer system
(Bad Homburg, Germany) using a preparative C18 column GROM-SIL 120 ODS-4 SE
with 10 µm silica, 250×20 mm, 120 Å pore size (Herrenberg-Kayh, Germany) and C4
DELTA-PAK 300×19 mm I.D., 300 Å (Nihon Water Ltd., Japan) or semipreparative
columns Phenomenex Jupiter C18 250×10 mm I.D. with 10 µm silica 300 Å pore size
(Torrance CA) and Vydac C4, 250×10 mm I.D. with 10 µm silica 300 Å pore size
(Hesperia CA). Chromatograms were recorded by UV detection at 220 nm.
Experimental part 108
3.4.2. Sample concentration and desalting using Zip Tip pipette tip
The Zip Tip pipette tip is a 10 µl pipette tip containing a bed of C18 or C4 reverse
phase media for desalting and concentration peptides and proteins.
The procedure requires the following solutions:
Wetting solution 50 % ACN in Milli-Q grade water
Washing and equilibration solution 0.1 % TFA
Elution solution 0.1 % TFA/ 50 % ACN
The lyophilised peptide sample is solubilised in 10-50 µl washing and equilibration
solution. If necessary, the pH of the final sample solution is adjusted to pH<4 with 1%
TFA. 10 µl of wetting solution are aspirated using a 10 µl pipettor and discarded to
waste. The step is repeated 4 times with wetting solution and 10 times with washing
and equilibration solution. To bind the peptides, the sample is aspirated and
dispensed in the reaction tube 10 to 20 cycles. The salts remained in the tip are
removed by 2 cycles of aspirating and discarding washing and equilibration solution
to waste. The peptides are eluted from the reverse phase media with 4 µl of elution
solution. During the procedure the aspiration of air bubbles has to be avoided.
3.4.3. One-dimensional gel electrophoresis
The separation of low-complexity protein mixtures was achieved by one-dimensional
gel electrophoresis carried on a Mini-Protean II or Mini-Protean 3 electrophoresis cell
(BioRad, München, Germany). The dimensions of the gel are 90 x 60 x 1 mm.
The proteins are solubilised and denatured using the stock solution of the two fold
concentrated sample buffer containing 4 % SDS, 25 % Glycerin, 50 mM Tris, 0.02 %
Coomassie, 6 M urea, pH 6.8. Reduction of the disulfide bridges is achieved by
addition to the stock solution of 50 times molar excess of DTT per cysteine residue
within the amino acid sequence of the protein. If the sample contains a mixture of
unknown proteins 100 mM DTT is added. The samples which were not subjected to
in gel-digestion of the protein spots subsequent to the 1D-gel separation, were
Experimental part 109
heated for 5 minutes at 56°C to ensure complete the denaturation. Protein samples
separated by electrophoresis followed by in gel digestion and mass spectrometric
molecular weight determination and sequence assignment of the fragments present
in the proteolytic mixture are not heated above 30°C to avoid protein carbamylation
and the stock solution of sample buffer should be stored in aliquots at -20°C. Urea in
water exists in equilibrium with ammonium cyanate, the level of which increases with
increasing temperature and pH. Cyanate reacts with the amino group of the Nterminus and ε-amino group of lysines. This reaction leads to artifactual charge and
molecular weight heterogeneity.
Regardless of the electrophoresis cell and method, preparation of the gel requires
casting of two different layers of acrylamide between glass plates. The separating gel
(lower layer) is responsible for the separation of the proteins according to their size.
The stacking gel (upper level) contains the sample wells and allows the concentration
of the sample at the limit between the two layers.
3.4.3.1. SDS-PAGE according to Laemmli
Proteins with molecular masses between 20 and 100 kDa were separated according
to the experimental procedure described by Laemmli. The following stock solutions
were used:
Acrylamide-bis: 29.22 % acrylamide, 0.78 % bisacrylamide (w/v)
4xStacking gel buffer 0.5 M Tris, 0.4% (w/v) SDS, pH 6.8
4xSeparating gel buffer 1.5 M Tris, 0.4% (w/v) SDS, pH 8.8
10xElectrode (Running) buffer 0.25 M Tris, 2M Glycin, 1% (w/v) SDS
APS 10 % (w/v) Ammonium peroxydisulfat in MilliQ
H2N PeptideH-N=C=O + H2N-C-NH Peptide
=
Isocyanic acid Peptide amino terminus
or side chain of Lys or Arg
O
NN
O
H2H2 NH4
+ + NCO-
Urea Ammonium
cyanate
Carbamylated peptide
or protein
heat, time
=
Figure 73: Schematic representation of protein carbamylation.
Experimental part 110
Table 13: Calculated volumes required for the preparation of 4 gels
Monomer concentration Stacking gel Separating gel Separating gel Separating gel
(%T, 2.6 % C) 5% 10% 12% 15%
4xStacking gel buffer 2.5 ml - - 4xSeparating gel buffer - 6 ml 6 ml 6 ml
MilliQ 5.8 ml 10 ml 8.4 ml 6 ml
Acrylamide-bis 1.7 ml 8 ml 9.6 ml 12 ml
TEMEDa 20 µl 20 µl 20 µl 20 µl
APS 85 µl 125 µl 125 µl 125 µl
a: N,N,N´,N-Tetramethylethylendiamin
3.4.3.2. SDS-PAGE according to Schägger and Jagow
The polyacrylamide gel electrophoresis approach described by Schägger and Jagow
[183] was employed for increased resolving power of proteins with molecular mass
below 10 kDa.
The stock solutions prepared for gel electrophoresis are given below:
Anode buffer 0.2 M Tris, pH 8.9
Cathode buffer 0.1 M Tris, 0.1 M Tricine, 0.1 % SDS
Gel buffer 3 M Tris, 0.3% SDS, pH 8.45
Table 14: Preparation of a 12% tricine-polyacrylamid-gel according to Schägger and Jagow
Monomer concentration Stacking gel Separating gel
(%T, 3 % C) 4 % 12 %
Gel buffer 3.1 ml 10 ml
Glycerin - 4 ml
30 % Acrylamide 1.94 ml 11.64 ml
2% Bisacrylamide 2.25 ml 5.4 ml
MilliQ 7.71 APS 75 µl 150 µl
TEMED 20 µl 15 µl
Gels were run at 60 V until the tracking dye entered the separating gel and at 120 V
until the tracking dye reached the bottom of the gel.
Experimental part 111
3.4.4. Two-dimensional gel electrophoresis
The separation of complex protein mixtures was achieved by two-dimensional gel
electrophoresis. The proteins are separated according to their charge by isoelectric
focusing (IEF) in the first dimension and according to size by SDS-PAGE in the
second dimension.
The protein mixtures to be separated were solubilized in a solution containing 7 M
urea, 2 M thiourea, 4% CHAPS, 0.3% DTT, 2% Servalyt 3-10 and a trace of
bromopheol blue. The total volume of sample used for rehydration is specified in the
manufacturer data sheet according to the length of the IPG strip and is a critical
parameter for a successful isoelectric focusing. The sample was pipetted in a slot of
the reswelling tray and the dehydrated IPG strip was placed in the slot with care to be
in contact with the solution in equal manner along the surface and not to trap air
bubbles under the gel. The solution was allowed to swell the gel for 20 min than 3 ml
of silicon oil were overlaid and allowed to stay overnight (12 h). For isoelectric
focusing [184-186] the IPG strips were removed from the rehydration tray, washed
with MilliQ and placed in the aligner of the flatbed Multiphor II unit. Electrode strips
moistened with MilliQ are placed above the cathodic and anodic ends of the aligned
IPG strips in contact with the gel. After aligning the electrodes over the electrode
strips, the IPG strip is overlayed with silicon oil to prevent dehydration and the
isoelectric focusing is run at 20° C using the voltage gradient described in the Table
15.
Table 15: Voltage gradient for the isoelectric focusing of rehydrated IPG strips
Phase Voltage (V) Duration (h:min) Voltage mode kVh
1 150 0:01 gradient 0.001
2 150 1 constant 0.15
3 300 0:30 gradient 0.11
4 300 2 constant 0.6
5 3500 2:30 gradient 4.7
6 3500 2-10* constant 7-35
Total 12.5-40.2
* The isoelectric focusing is finished when the current remains constant for 30 min
Experimental part 112
After isoelectric focusing the IPG strips were equilibrated for 30 min in 6 M urea, 30%
glycerol, 2% w/v SDS, 0.05 M Tris-HCl (pH 8.8), 1% w/v DTT, 0.002% bromophenol
blue, then for 30 min in the same solution except that DTT was replaced by 4.5%
(w/v) iodoacetamide. The excess of buffer on the strips was removed by blotting on a
filter paper. The separation in the second dimension was carried out using a
PROTEAN II xi cell. The strips were placed orizontally on a 190 x 190 x 1.5 mm SDS
PAGE separating gel prepared according to the details from Table 13.
3.4.5. Colloidal Coomassie staining
Stock solutions:
Fixing solution 12% (w/v) trichloroacetic acid
A 10% (w/v) ammonium sulphate, 2% phosphoric acid
B 5 % (w/v) Coomassie Brilliant Blue G-250
Washing solution 25 % methanol
The gel is incubated for 1 h in 100 ml fixing solution and for 12 h in staining solution.
The staining solution is prepared prior to use, by mixing for 30 min 80 ml of the stock
solution A with 2 ml stock solution B. 20 ml of methanol p.a. are added and the
solution is mixed for 30 min. After staining, the gel is washed for 5 and 30 min with
25 % methanol [187].
3.4.6. Silver staining
Stock solutions:
Fixing solution 30 % (v/v) Ethanol, 10 % (v/v) acetic acid
Sensitizing solution 11.2 % (w/v) Sodium acetate trihydrate, 30 % (v/v) ethanol
Prior to use: 0.2 % (w/v) sodium thiosulphate-5-hydrate
Staining solution 0.2 % (w/v) Silver nitrate
Prior to use: 0.0074 % formaldehyde
Developing solution 2.5 % Sodium carbonate
Prior to use: 0.0037 % formaldehyde
Formaldehyde 37 % Formaldehyde
Experimental part 113
After SDS-PAGE, the gel was incubated between 30 and 60 min in fixing solution
and 30 min or overnight in sensitizing solution. After 3 washing steps with MilliQ (5
min) the gel was incubated in staining solution for 20 min. The solution was
discarded and the developing solution was added in 2 steps: 1 min then discarded
and 5-20 min until the desired ratio of spot intensity to background was observed.
3.5. Immunological methods
3.5.1. Preparation of immobilised antibodies
Antigen specific antibodies were dissolved in 0.2 mol L-1 NaHCO3, 0.5 mol L
-1 NaCl
coupling buffer (pH 8.3) to a final concentration of 0.5-1 µg/µl. The solution was
added to dry NHS-activated 6-aminohexanoic acid-coupled Sepharose (Sigma, FRG)
and the coupling reaction was performed 1h at 20ºC. The Sepharose-coupling
product was loaded into a 0.8 mL micro-column (Mobitec, Göttingen, FRG) and
washed sequentially with blocking (0.1 mol L-1 aminoethanol, 0.5 mol L-1 NaCl, pH
8.3) and washing solution (0.2 mol L-1 CH3COONa 0.5 mol L
-1 NaCl, pH 4.0). Each
solvent was used with a total volume of 24 ml (four times 6 ml) and the procedure
was repeated after 1h incubation in blocking buffer. For long time storage at 4 °C, 1
mmol L-1 Na2HPO4, 136 mmol L-1 NaCl, 2.7 mmol L-1 KCl and 0.01% NaN3 (pH 7.3)
was used.
3.5.2. Epitope excision and extraction experiments
For epitope excision experiments the antigen was dissolved in PBS containing 5 mM
Na2HPO4, 150 mM NaCl, pH 7.5 and applied to the affinity matrix considering an
immobilized antibody:antigen ratio of 2:1 to avoid non-specific binding. The microcolumn was gently shaken for 2h to allow complete binding of the antigen. Nonbound peptide was removed by blowing out the column almost to complete dryness
of the affinity matrix using a 10 mL syringe and subsequently washing with 10 mL
PBS. Remaining affinity-bound antigen was digested for 2h at 37°C with 1 µg TPCKtreated trypsin in 50 mM NH4HCO3 or 20h with endoprotease Glu-C. Supernatant
non-epitope containing peptides were removed again by blowing out the column with
Experimental part 114
a syringe, and the material was washed with 10-50 ml PBS buffer. The appropriate
amount of PBS necessary for washing is established in a preliminary experiment in
order to ensure complete removal of unspecifically bound peptide fragments before
eluting the epitope peptides. The immune complex was dissociated by addition of
500 µl 0.1% trifluoroacetic acid. The micro-column was gently shaken 15 min at 20°C
and the released epitope peptides collected in a microreaction cup. A second elution
is performed if the pH of the previous elution fraction exceeds the value 3.5. The
column was regenerated by washing with 10 ml 0.1 % trifluoroacetic acid followed by
20 ml PBS buffer. Epitope extraction was performed by appling the proteolytic
mixture directly to the column.
3.5.3. Preparation of immobilized antigen column
Due to the low solubility of the Cys-Aß(1-40), 3 mg of peptide were dissolved in 50
mM Tris, 5 mM EDTA-Na coupling buffer (pH 8.5) to a final concentration of 3 mg/ml.
The solution was added to 1 ml of drained Ultralink Iodoacetyl Gel and the coupling
reaction was performed at room temperature for 30 minutes under gentle mixing
followed by 30 minutes without mixing. The matrix-coupling product was loaded into
a 2.5 ml column allowing the solution to drain. The column was washed with 3 ml of
coupling buffer and the non-specific binding sites on gel were blocked 2 times for 45
minutes with 1 ml of 50 mM L-Cysteine•HCl in coupling buffer. At the end, the column
was washed with 5 ml of 1M NaCl and 5 ml of 0.1M phosphate, 0.15M NaCl (pH 7.2)
and stored at 4°C.
3.5.4. Separation of Aß-autoantibodies from pooled IgG preparations
Since Aß(1-40) contains two internal lysine residues an improved affinity column
purification protocol was developed using a cysteine attached to the N-terminus of
the compound. This ensures that every peptide molecule will be oriented on the
column support in the same way after immobilisation. The azlactone-activated
support contains an iodoacetyl group (UltraLink; Perbio, Bonn, Germany) at the end
of a 15 atom long spacer arm, the former to react with a sulfhydryl group to produce
Experimental part 115
a stable thioether linkage, the latter to reduce steric hindrance and thus providing
enhanced binding of the specific antibodies.
0.5 ml of the Cys-Aß(1-40) coupled support was packed into a column (2.5 ml,
MoBiTec, Göttingen, Germany) and equilibrated with 20 ml of PBS (0.1M sodiumphosphate, 0.15M NaCl, pH 7.2). The support was transferred into a 15 ml Falcon
vial using 5 ml PBS and mixed with 5 ml IVIgG (Bayer Vital GmbH, Leverkusen). The
diluted gel slurry was slowly rotated overnight at 4°C. The suspension was
transferred to a column using the effluent to completely rinse the matrix back into the
column. The column was washed four times with 10 ml of PBS followed by 4 wash
cycles with 10 ml 5 mM Na2HPO4, 150 mM NaCl, pH 6.8 and two wash cycles with
MilliQ.
The affinity-bound antibodies were eluted from the column with 10x0.5 ml 0.1% TFA.
IgG preparation for the following experiments was performed using two different
protocols. The first procedure involved adjusting neutral pH for each fraction
collected using 0.5M NaH2PO4, pH 8.9 in order to maintain integrity of the antibodies
used in affinity tests. For 2D-PAGE the elution of the specifically bound IgG was not
accompanied by any pH adjustment in order to reduce the salt content present in the
sample subjected to isoelectric focusing. To regenerate the column, the matrix was
washed with 10 ml TFA and 40 ml of PBS.
3.5.5. Enzyme-linked immunosorbent assay
3.5.5.1. Alanine scanning mutagenesis
All assays were performed in 96-well plates coated over night at 4ºC with 150 µL/well
of 5mg/l streptavidin. Wells were washed one time with 0.05 % v / v Tween 20 in
PBS (Na2HPO4 5mM, NaCl 150mM, pH 7.5). Peptide samples were prepared and
diluted in PBS to a final concentration of 0.5 µM. 100 µL of each solution were
deposited in the wells in triplicate and the plates were incubated for 2 hours at room
temperature. For background substraction, triplicate wells containing PBS have been
incubated in the first step for each antibody dilution. The wells were blocked with
BSA 5 % w / v in PBS for 2 hours, and washed with PBS / 0.05 % v / v Tween 20.
Experimental part 116
The monoclonal antibodies (mAb) 6E10 from Chemicon (Temecula, CA; raised
against Aß(1-17), purified IgG) or polyclonal IgGs from Aß42 immunized TgCRND8
mice were added in 8 two fold serial dilutions and the 96-well plates were gently
shaken for 2h to allow complete binding of the antibody. Supernatant non-bound
antibodies were removed by four times washing-step with PBS / 0.05 % v / v Tween
20. Alkaline phosphatase-conjugated goat anti-mouse antibody (Sigma, Saint Louis,
USA; 100 µL at 1 µg / mL in BSA 5 % w / v in PBS) was introduced and incubated for
2 hours at room temperature. The wells were washed twice with PBS / 0.05 % v / v
Tween 20, and twice with diethanolamine 2.5M, pH 9.5. Wells were finally incubated
for 1-30 minutes at room temperature with Attophos substrate (Promega, Madison,
USA) and the enzyme reaction was monitored as a function of time with a 485/535
(excitation/emission) filter set, using an ELISA plate reader (Victor2, Perkin Elmer
Life/Analytical Sciences, Boston, MA).
Figure 74: Schematic representation of indirect ELISA.
Coating the wells
with streptavidin
Binding the
biotinylated peptides
Blocking with
5% BSA
Binding the antigen
specific antibody
Binding the
detection antibody
Streptavidin
Peptide derivatized with a pentaglycine spacer
and biotin at the amino-teminus
BSA
Antigen-specific
antibody
Detection antibody
Experimental part 117
3.5.5.2. Zinc binding effects on the antigen-antibody interaction
All assays were performed in 96-well plates coated over 1 night at room temperature
with 100 µL of the monoclonal antibodies (mAb) 6E10 or Bam 10 at 1 µg / mL IgG in
bicarbonate buffer (pH 9.6). Wells were washed four times with 0.05 % v / v Tween
20 in TB (Tris-(hydroxymethyl)aminomethan / HCl buffer, 10 mM, pH 7.5). The wells
were blocked with BSA 5 % w / v in TB for 2 hours, and washed with TB / 0.05 % v /
v Tween 20. Peptide samples were prepared and diluted in TB. All metal ions were
subsequently added under the form of chloride salts. For any desired final
concentration of metal ions, 10 µL of a stock solution freshly prepared in water were
added to 330 µL of the peptide solution. 100 µL of each solution were deposited in
the wells in triplicate and the plates were incubated for 2 hours at room temperature.
The wells were washed four times with TB / 0.05 % v / v Tween 20. 100 µL of
horseradish peroxidase-conjugated goat anti-biotin antibody (1 µg / mL in BSA 5 %
w / v in TB) was added and incubated for 2 hours at room temperature. The wells
were washed twice with TB / 0.05 % v / v Tween 20, and twice with citrate phosphate
buffer, pH 5.0. Wells were finally incubated for 10-30 minutes at room temperature
with o-phenylenediamine [188-190] dissolved in citrate phosphate buffer, pH 5.0, and
the enzyme reaction was monitored as a function of time at 450 nm using an ELISA
plate reader (Victor2, Perkin Elmer Life/Analytical Sciences, Boston, MA, USA).
S
N S
N
O
P
O- O-
O
S
N S
N
O-
-Pi
AP, pH9-10.3
2´-[2-benzothiazoyl]-6´-hydroxybenzothiazole
phosphate
2´-[2-benzothiazoyl]-6´-hydroxybenzothiazole
Figure 75:The reaction of Attophos substrate (2´- [2-benzothiazoyl]-6´-hydroxybenzothiazole
phosphate) to produce the fluorescent emitter (2´-[2-benzothiazoyl]-6´hydroxybenzothiazole) when an alkaline phosphatase labeled antibody liberates the
phosphate group.
Experimental part 118
Figure 77: Schematic representation of sandwich ELISA.
Coating the wells
with Aß-specific
antibody
Blocking with
5% BSA
Binding the biotinylated
antigen to the specific
antibody
Binding the anti-biotin
detection antibody
Peptide derivatized with a pentaglycine spacer
and biotin at the amino-teminus
BSA
Antigen-specific antibody
Detection antibody
NH2
NH2
N
N
NH2
NH2
2H2O2 + 2
HRP, pH 5.0
o-phenylenediamine
(OPD)
2,3-diaminophenazine
(OPD)
Figure 76: Schematic representation of the reaction catalysed by horseradish peroxidase.
The oxidation product of o-phenylenediamine produced by horseradish peroxidase
is 2,3-diaminophenazine. This product is orange-brown in color and can be read
spectrophotometrically at 450 nm.
Experimental part 119
3.6. Chemical modification reactions and enzymatic fragmentation of
proteins
3.6.1. Reduction and alkylation of disulfide bonds in solution
Disulfide bridge reduction is achieved by addition of 50 times molar excess of DTT
per cysteine residue followed by incubation at 56°C for 1 h. The sample is alkylated
by incubation for 1h at room temperature and dark with 2.5 molar excess of IAA over
DTT.
Carboxymethylation may be carried out without prior reduction in order to modify only
the cysteine residues that are not involved in disulfide bridges.
3.6.2. Proteolytic digestion of proteins in solution using trypsin
Peptides and proteins to be proteolytically digested were solubilised in 10 mM
NH4HCO3, pH 8, at concentrations of 0.1-1 µg/µl. Alternatively enzymatic digestion
was carried out in phosphate buffer saline containing 5 mM Na2HPO4, 150 mM NaCl,
pH 7.5 and in 50 mM Tris-HCl, 20 mM CaCl2 pH 7.8. Stock solutions of the sequence
grade TPCK-modified porcine trypsin 1 µg/µl in 50 mM acetic acid were stored at 20°C. Trypsin was added to the sample at an enzyme to substrate ratio of 1:20 (w/w)
and the digestion was carried out at 37°C. The reaction was monitored in time by
removal of sample aliquots, quenching by freezing in liquid nitrogen and subsequent
mass spectrometric analysis of the proteolytical peptides [191].
3.6.3. Proteolytic digestion of proteins in solution using endoproteinase GluC
To prepare stock solutions of endoproteinase GluC, lyophilized preparation was
reconstituted in MilliQ to a final concentration of 1 µg/µl. The stock solution was
stored at -20°C. Digestion of peptides was performed in PBS containing 5 mM
Na2HPO4, 150 mM NaCl, pH 7.8. An enzyme:substrate ratio of 1:20 was employed.
The incubation time was between 4 and 20 hours at 37°C.
Experimental part 120
Table 16: Peptide sequence assignment of the molecular ions present in the supernatant of the
epitope extraction from the proteolytic mixture produced by enzymatic digestion with GluC
for 20 hrs
[M+H]+exp [M+H]
+
calc. ∆m
Da Proteolytic
fragment
Sequence
574.6 574.6 0 [4-7] 4FRHD7
1010.4 1011.0 0.6 [4-11] 4FRHDSGYE11
1326.0 1326.3 0.3 [1-11] 1DAEFRHDSGYE11
1355.7 1355.5 0.2 [12-22] 12VHHQKLVFFAE22
1566.9 1570.6 3.7 [24-40] 24VGSNKGAIIGLMVGGVV40
1686.6 1686.0 0.6 [23-40] 23DVGSNKGAIIGLMVGGVV40
2349.3 2347.6 1.7 [4-22] 4FRHDSGYEVHHQKLVFFAE22
2777.6 2777.9 0.3 [1-23] 1DAEFRHDSGYE VHHQKLVFFAED22
3025.1 3022.6 2.5 [12-40] 12VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
Table 17: Sequence assignment of molecular ions observed in the supernant fraction from the epitope
extraction using GluC
[M+H]+exp [M+H]
+
calc. ∆m
ppm
Proteolytic
fragment
Sequence
1010.4303 1010.4327 2 [4-11] 4FRHDSGYE11
1210.5113 1210.5124 1 [2-11] 2 AEFRHDSGYE11
1325.5340 1325.5393 4 [1-11] 1DAEFRHDSGYE11
1684.9410 1684.9415 0 [23-40] 23VGSNKGAIIGLMVGGVV40
3020.6706 3020.6503 7 [12-40] 12VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
4012.1071 4012.0651 10 [4-40] 4FRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
4326.9600 4327.1717 49 [1-40] 1DAEFRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
3.6.4. Proteolytic digestion of proteins in solution using alpha-chymotrypsin
Stock solutions of alpha-chymotrypsin 1 µg/µl were prepared in 1mM HCl containing
2 mM CaCl2. The solution was stored at -20°C as frozen aliquots for 1 week. For
peptide digestion a ratio of 1:20 (w/w) chymotrypsin: peptide was used. The digestion
was performed for 20 hours, at 37ºC in PBS containing 5 mM Na2HPO4, 150 mM
NaCl, pH 7.5.
Experimental part 121
Table 18: Peptide sequence assignment of the molecular ions present in the proteolytic mixture
produced by digestion of Aß(1-40) with alpha-chymotrypsin.
[M+H]+exp [M+H]
+
calc. ∆m
Da Proteolytic
fragment
Sequence
734.2 734.7 0.5 [5-10] 5RHDSGY10
803.6 803.0 0.6 [28-35] 28KGAIIGLM35
890.2 891.1 0.8 [11-17] 11EVHHQKL17
1099.3 1100.1 0.8 [18-27] 18VFFAEDVGSN27
1477.5 1477.6 0.1 [21-35] 21AEDVGSNKGAIIGLM35
1606.7 1606.7 0 [5-17] 5RHDSGY EVHHQKL17
1856.5 1853.0 3.5 [5-19] 5RHDSGY EVHHQKLVF19
3.6.5. Proteolytic digestion of proteins in solution using Pronase
The enzyme is supplied as lyophilized powder. Stock solution of 10 µg/µl were
prepared in MilliQ and stored at 4°C for maximum one week. Pronase digestions
were performed at 40 ºC, in PBS containing 5 mM Na2HPO4, 150 mM NaCl, pH 7.5.
An enzyme:substrate ratio of 1:1 (w/w) and concentrations of 0.1 µg/µl for Aß(1-40)
and 0.5 µg/µl for pronase were used. The Aß(1-40) was diluted to 0.1 µg/µl due to the
tendency to aggregate at higher concentrations.
3.6.6. Proteolytic digestion of proteins in solution using Endoproteinase
LysC
Lyophilizate Endoproteinase Lys-C was reconstituted in 50 µl MilliQ. This results in a
solution containing 0.1 µg/µl enzyme, 50 mM HEPES, pH 8.0, 10 mM EDTA and 5
µg/µl raffinose. The digestion was carried out at 37°C, in 25 mM Tris HCl, pH 8.5,
containing 1mM EDTA for 5 hours. The amount of enzyme was 1:20 of the protein by
weight.
Experimental part 122
3.6.7. In-gel trypsin digestion procedure of Coomassie Brilliant Blue stained
proteins
Before excising the bands, the gel was washed in MilliQ for 15 min. The spot of
interest was excised from the gel, cut into 1 mm2 pieces and placed in a sample tube.
After washing for 15 min with 100 µl MilliQ, the liquid was discarded and the gel
pieces were dehydrated by incubation for 30 min in 100 µl of 60% ACN. The liquid
was discarded and the gel pieces were dried using a centrifugal vacuum evaporator.
The pieces are rehydrated with 100 µl 50 mM NH4HCO3 for 15 min and dehydrated
with 60% ACN. This step is repeated until the gel pieces are destained. The gel
pieces are lyophilized to complete dryness. For digestion, an enzyme solution
containing 50 mM NH4HCO3 and 12.5 ng/µl trypsin is prepared on ice. The gel pieces
are allowed to rehydrate for 45 min on ice, in the appropriate volume of enzyme
solution needed to completely swell the gel. The excess of enzyme solution is
removed, the gel pieces are covered with 50 mM NH4HCO3 and incubated overnight
at 37°C. Proteolytic peptides are extracted by incubating gel pieces with ACN/0.1%
TFA (3/2) for 1h at room temperature. The solution is collected and the step is
repeated 2 times.
3.7. Mass spectrometric methods
3.7.1. Time of flight mass spectrometry
The principle and design of the time-of-flight mass spectrometers are among the
simplest of the mass analysers. The technique involves mixing the analyte of interest
with a large molar excess of a matrix compound, usually a weak organic acid. The
sample target is inserted into the time-of-flight mass spectrometer. The analyte is
desorbed into the gas phase by irradiation with short laser pulses. The charged
analyte is accelerated to a fixed kinetic energy by an electric potential (U). Mass-tocharge ratios are determined by measuring the time (t) it takes for ions to move in a
field-free region of length l. A detector placed at the end of the flight tube produces a
signal as each particular packet of ion species strikes it. Given a constant
accelerating voltage, the flight time of an ion is related to its m/z ratio [192, 193].
Experimental part 123
Sample preparation is a crucial procedure in the matrix assisted laser desorption and
includes removal of contaminants such as salts, detergents, denaturants and the cocrystallization with the matrix on the sample target. Desalting and concentration of
the sample was achieved by using the ZipTip C18 pipette tips as described before.
Samples were loaded onto a 26-spot target plate using the dried-droplet method
[194] : 0.7 µl of matrix solution (saturated solution of HCCA in acetonitrile:0.1% TFA
2:1 v/v) were loaded onto the sample plate and 0.7 µl of sample solution were
deposited on the matrix droplet, mixed and allowed to dry in the ambient air.
However this procedure was shown to result in considerable oxidation of methionine
and tryptophan.
Mass spectra were recorded using a Bruker Biflex II linear MALDI-TOF mass
spectrometer (Bruker Daltonik, Bremen, Germany), equipped with a pulsed nitrogen
laser (337 nm). The instrument is operated in delayed extraction mode with an
acceleration voltage of 20 kV.
To ensure accurate molecular weight determination, the instrument was calibrated
prior to each measurement using the external calibration method. A mass spectrum
was aquired for a mixture of standards consisting of human angiotensin II (1047.2),
human bradykinin (1061.2), human angiotensin I (1297.2), human
adrenocorticotropic hormone (ACTH) (2466.7), bovine insulin B-chain, oxidized
(3496.9), bovine insulin (5734.5). The calibration constants are automatically
Laser
shot
+
+
+
+
+
+
+
Extraction and
acceleration grids
Flight tube
+
+
+
++
+
+
++
+
Detector
lU Acceleration region Field free drift region
+
Figure 78: Schematic representation of a linear MALDI-TOF mass spectrometer. The analytes are
separated in the flight-tube based on their masses. The lighter molecules reach the
detector faster than the heavier molecules
Experimental part 124
calculated by a linear regression fit after assignment of the peaks in the mass
spectrum using the reference masses.
3.7.2. Fourier-transform Ion-Cyclotron Resonance mass spectrometry
FT-ICR mass spectrometric analysis was performed with a Bruker APEX II FT-ICR
instrument equipped with an actively shielded 7T superconducting magnet (Magnex,
Oxford, UK), a cylindrical infinity ICR cell. The ICR cell consists of 3 opposing pair of
plates placed in the homogeneous region of the magnet. The analyte ions are
trapped inside the cell by a low potential (1V) applied to the trapping plates. In a
strong magnetic field along the z-direction the ions are constrained to move in
circular orbits in the xy-plane referred as cyclotron motion. The frequency of the ion
cyclotron motion is identical for all ions of the same m/z and is independent of their
velocity as it can be seen in the cyclotron equation:
ω = zB/m
The excitation is accomplished by applying an alternating voltage to one opposing
pair of plates oriented parallel to the electric field. If the frequency of the alternating
electric field equals the cyclotron frequency of the ions of a particular m/z, then the
cyclotron motion of these ions will be coherently excited. After excitation these ions
move as a single ion packet on an orbit with increased radius.
The rotating ion packet induces an alternating charge in the detection plates of an
ICR cell and an alternating voltage across this conductor, which is amplified and
B
x y z Excite
Detect
Figure 79: Schematic representation of an ICR cell used in FTMS.
Experimental part 125
registered as transient or time domain signal. After its acquisition, the time domain
data is stored and subsequently Fourier transformed yielding the cyclotron
frequencyspectrum, which is converted to the mass soectrum. A very good vacuum
(10-9) is required to avoid collisions of the ion packet in the cell with neutral molecules
that could lead to a decrease of the radius.
3.7.2.1. MALDI-FT-ICR mass spectrometry
MALDI-FT-ICR mass spectrometry was performed with the Bruker APEX II FT-ICR
mass spectrometer using and an external Scout 100 fully automated X-Y target stage
MALDI source with pulsed collision gas [195, 196]. A schematic representation of the
APEX II FT-ICR mass spectrometer equipped with MALDI source is depicted in the
Figure 80. The MALDI target is a circular steel plate with 25 or 49 sample placement
spots. This plate is placed on the end of a cylindrical target manipulation rod and held
in place by a magnet. The rod can be inserted into or removed from the mass
spectrometric vacuum system. In the inserted position, the target plate is fixed at 1
mm distance from the hexapole ion guide. The pulsed nitrogen laser was operated at
337 nm and ions generated in the MALDI process were cooled with the pulsing
collision gas and directly captured into the hexapole ion guide. Ions generated by 2030 laser shots were accumulated in the hexapole for 0.5-1s at 30V and extracted at 15V.
Figure 80: Schematic representation of Bruker APEX II FT-ICR mass spectrometer equipped with
MALDI source.
Scout 100 MALDI source Superconducting
magnet
ICR cell
Ion transfer optics
Collision
gas tube
Sample
target
Laser beam
Gate valve
500 L/sec
Turbo pump
10-10 mbar
70 L/sec
Turbo pump
10-8 mbar
500 L/sec
Turbo pump
10-6 mbar
Hexapole
Extraction and
trapping plate
Experimental part 126
A 100 mg/ml solution of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/0.1% TFA in
water (2:1 v/v) was used as matrix. The analyte was dissolved in the same solvent.
0.5 µl of matrix solution were depozited on the stainless steel MALDI target, mixed
with 0.5 µl of sample solution and allowed to dry in ambient air. Mass spectra were
obtained using 10 laser shots for each scan and accumulating 32-64 scans. External
calibration was performed prior to each measurement using the monoisotopic
masses of singly protonated ion signals of human angiotensin II (1046.542), human
bradykinin (1060.569), human angiotensin I (1296.685), human neurotensin
(1672.917), bovine insulin ß-chain oxidized (3494.651) and bovine insulin (5730.609).
3.7.2.2. ESI-FT-ICR mass spectrometry
ESI-FT-ICR mass analysis was performed with a Bruker APEX II FT-ICR mass
spectrometer equipped with the APOLLOTM API ionisation source. The sample was
dissolved in a solvent containing 50% methanol, 48% water and 2% acetic acid. The
sample solution was introduced through a PEEK capillary to the spraying needle
using a syringe pump with a flow rate of 2 µl min-1. A voltage of -4200 V was applied
between the metal coated entry of the glass capillary and the grounded spray needle.
The voltage applied on the endcap was -3800 V. A nebulizing gas (N2) was employed
to stabilise the spray. Desolvation was facilitated by using a drying gas (N2) heated at
150°C. Before each measurement cycle, the source and the analyser cell were
quenched for 50 msec. The ions produced by the ESI process passed through the
glass capillary and from the capillary exit they streamed through the skimmer
entering into the hexapole ion guide. The declustering potential (CS), which is the
small potential difference between the capillary exit and the skimmer, was set to 6070 V. After a predefined accumulation time in the hexapole (0.1-2 sec) the voltage of
the extraction plate was reversed and the ions were extracted for transmission to the
ICR cell. External calibration was carried out using monoisotopic masses of
angiotensin I fragment ions formed by in-source fragmentation (CS=150 V).
Experimental part 127
3.7.3. Liquid chromatographic/Ion trap mass spectrometric investigation
For LC/MS investigations, an Agilent Technologies (Waldbronn, Germany) HP1100
liquid chromatograph for binary gradient elution (pump model G1312A), including
autosampler (G1313A) and a DAD (G1315 B), coupled to an Esquire 3000 ion trap
mass spectrometer from Bruker Daltonics (Bremen, Germany) was used. For the LC
separation, a binary gradient system consisting of solvent A (0.1% formic acid in
water) and solvent B (80% acetonitrile, 0.1% formic acid in water) was employed.
The sample was dissolved in the solvent A. The injection volume was 5 µl. A 150 mm
x 4.6 mm x 3 µm Discovery RP-18 column was used for the separation of the
peptides.
All LC/MS results were obtained using atmospheric pressure chemical ionization
(APCI) in the positive ion mode. Mass spectra were recorded in the full scan mode,
scanning from m/z 200 to 1500. Ion source parameters were 20 psi nebulizer gas
and 10.0 L/min of drying gas with a temperature of 300 °C. MS/MS experiments were
carried out in the autofragmentation mode.
APOLLOTM API source Superconducting
magnet
ICR cell
Ion transfer optics
Collision
gas tube
Laser beam
Gate valve
500 L/sec
Turbo pump
10-10 mbar
70 L/sec
Turbo pump
10-8 mbar
500 L/sec
Turbo pump
10-6 mbar
250 L/sec
Turbo pump
10-4 mbar
Extract
and trap
Extraction and
trapping plate
5 L/sec
Rotary vane
10-1 mbar
Hex ole
Cylinder
Capillary
± 4200
ap llary
exit
Skimmer
Nebulising gas
Drying
gas
Figure 81: Schematic representation of the Bruker APEX II FT-ICR mass spectrometer equipped with
APOLLO ESI source
Experimental part 128
3.8. Bioinformatic tools for mass spectrometry
3.8.1. GPMAW
General Protein/Mass Analysis for Windows (GPMAW) version 7.0 (Lighthouse Data,
Denmark) [154] was employed in this work for the analysis of intact and
proteolytically digested proteins with known sequence. The program enables
manually editing of peptide sequences as well as the import of a sequence from a
number of different formats with direct database search. The most common formats
are FastA format, Swiss-Prot and Genbank. Sequences can be saved in local files for
future reference.
Using this program the protein can be cleaved by automatic pre-defined methods or
manually. Cross-links and chemical modifications can be entered in GPMAW and
saved with the sequence. The peptides are displayed with a number of parameters
including monoisotopic and average mass of single and multiply charged ions with 2
Figure 82: Schematic representation of the Bruker Esquire 3000 ion trap mass spectrometer.
Capillary
Skimmer Partition
Sample inlet
Nebulizer
gas
Drying gas
Nebulizer
Waste
Two octopoles
Ring electrodes
End plates
Focus
lens
Electron
multiplier
Ion trap DetectorIon transport and focusingIon generation
Experimental part 129
and 4 decimals, HPLC index, pI. A list containing the molecular masses of all MS/MS
fragment ions can be invoked for the desired sequence. GPMAW may predict the
secondary structure, the hydrophobicity and the antigenic sequences of a given
protein or peptide.
3.8.2. Search engines for identifying proteins
Most of the identification and characterization programs compare user-submitted
data with information archived in one or more databases. The identification and
characterization of the proteins separated on 2D-gel and visualized by Coommassie
blue staining was achieved by proteolytic digestion of the proteins using trypsin,
mass spectrometric molecular weight determination of the fragments resulted and
sequence assignment by database search [197].
The search was carried out within the NCBInr protein database using the MASCOT
search engine available from Expert Protein Analysis System World Wide Web
server [198]. NCBInr is a comprehensive, non-identical protein database. The entries
have been compiled from a variety of sources including GenBank (National Institutes
of Health genetic-sequence database), PIR (International Protein Sequence
Database), SWISS-Prot, PRF (Protein Research Foundation) and PDB (Brookhaven
Protein Data Bank).
MASCOT search options against NCBInr database were 1 missing tryptic cleavage,
monoisotopic mass search with error tolerance between 5 and 30 ppm,
carbamidomethylcysteine as fixed modification, oxidized methionine and deamidation
(NQ) as variable modifications.
3.8.3. BALLView 1.1.1
BALLView is an open source software [11] employed in this work for the visualization
and modeling of molecular structures. The program is able to import structures from
the most common molecular structure formats (PDB, MOL, MOL2 and SD).
Experimental part 130
Structures can be visualized with all standard graphical models and coloring methods.
Different parts of a molecule can be freely visualized and selected for special tasks.
3.8.4. PDQuest 2-D gel analysis software
PDQuest image analysis software from Bio-Rad was used for imaging the 1D and
2D-gels and removal of the background noise and gel artifacts from the image.
Summary 131
4. SUMMARY
Recent advances in immunology and molecular biology have lead to the
development of therapeutic vaccines which are of potential use in chronic diseases
such as cancer, cardiovascular disorders and neurodegenerative diseases, where
efficacies of available therapies are poor. Future advances in vaccine development
will rely substantially on a more complete understanding of the structural basis of
immune response. Mass spectrometry has emerged as a widespread technique for
the study of protein structure, function and interaction with other biomolecules.
Important features of the mass spectrometric protein analysis are the high sensitivity,
high mass accuracy, short analysis time and low sample consumption. To obtain
information on complex protein mixtures and to dissect the structure of the molecular
recognition domains diverse applications have been developed in conjunction with
mass spectrometry. These methods include chromatographic and electrophoretic
separations, proteolytic assays, differential chemical modification of specific amino
acid functions and bioinformatic tools for data analysis.
One of the hallmarks of Alzheimer´s Disease is the accumulation in the human brain
of extracellular plaques containing aggregates of the neurotoxic ß-amyloid peptide.
The immunotherapeutical approaches capable of triggering the clearance of amyloid
plaques and preventing Aß aggregation have gained increasing interest in recent
years. The first two parts of the thesis are focused on the development and
application of mass spectrometric and immuno-analytical methods to the
identification of epitopes on Aß recognized by anti-Aß antibodies.
The first part of the thesis was focused on the detailed characterization of the ßamyloid (4-10) FRHDSGY interaction with cognate antibodies. The sequence has
been previously identified as a structural epitope for two antibodies, a polyclonal antiAß(1-42) and a monoclonal anti-Aß(1-17) antibody. In order to determine the
functional significance of these residues to the antibodies, site-directed mutagenesis
was performed using synthetic ß-amyloid (4-10) mutants as model substrate peptides.
Selective identification of the affinity preserving mutant peptides was achieved by
comparative ELISA binding studies. While the interaction to the polyclonal antibody
Summary 132
was preserved in the D7A, S8A, G8A and Y10A mutants indicating F4, R5 and H6 as
essential residues, for the monoclonal antibody all amino acid residues were
essential for binding. The binding of Aß(1-16) to mAb- anti(1-17), clone 6E10 and
mAb-anti(1-40) clone Bam-10 was studied in the presence of Zn2+ ions. Previous
mass spectrometric studies mapped Zn2+ binding sites to His6, His13 and His14.
Aß(1-10) and Aß(1-16) were synthesized by solid phase peptide synthesis according
to the Fmoc strategy with a pentaglycine spacer and biotin at the N-terminal end.
Both peptides reacted in a dose-dependent manner with the 6E10 and Bam-10 mAbs.
The presence of Zn2+ triggered a 4- and 10-fold increase in ELISA response of biotinG5-Aß(1-16) to both mAb 6E10 and Bam-10. By contrast, the presence of Zn
2+ was
without effect on the ELISA response of biotin-G5-Aß(1-10). The cations Co
2+ and
Ni2+ had no effect on biotin-G5-Aß(1-16) recognition. The presence of Cu
2+ ions did
not influence the recognition of biotin-G5-Aß(1-16) by 6E10 mAb but resulted in a
higher ELISA response with Bam-10. These results show a zinc induced
conformational effect on the N-terminal region Aß(1-16) of the ß-amyloid peptide
which may result in an enhanced accessibility of the F4-Y10 epitope to anti-Aß
antibodies.
The second and major part of the thesis was focused on the identification of the
epitope recognized by anti-Aß-autoantibodies naturally occurring in human blood.
The antibodies were isolated by affinity chromatography from human immunoglobulin
preparations, and serum samples of Alzheimer´s disease patients. An affinity column
for antibody isolation was prepared by immobilising Cys-Aß(1-40) on a iodoacetylsupport. For mass spectrometric epitope identification, an affinity column was
prepared using the purified antibodies. In epitope excision, selective proteolytic
cleavage of the intact Aß affinity- bound to the immobilised antibody was performed
using trypsin or endoproteinase V8, followed by MALDI-TOF mass spectrometric
analysis of the epitope- and non-epitope fractions, and provided direct information
that the epitope is located within the sequence Aß(12-40). A consistent result was
obtained by epitope extraction-mass spectrometry. The use of pronase provided the
identification of Aß(21-37) as the minimal epitope structure for recognition.
Comparative binding studies of human Aß-antibodies with Aß(1-16), Aß(1-40),
Aß(12-40) and Aß(17-28), each synthesized with a pentaglycine spacer and biotin at
the N-terminal end, were performed by indirect ELISA. The results showed that
Summary 133
Aß(1-16) and Aß(17-28) do not interact with the antibodies while Aß(12-40) and
Aß(1-40) reacted with the autoantibodies in a concentration-dependent manner.
Similar results were obtained by analyzing samples of anti-Aß autoantibodies
isolated from AD patients. The separation by 2D gel electrophoresis revealed the
polyclonality of the antibodies. MALDI-FT-ICR mass spectrometric analysis of the
tryptic mixture resulting from in-gel digestion of the protein spots led to the
identification of γ1 and γ2-heavy chains, and κ-light chains.
A further part of the dissertation was focused on the serine protease HtrA1 which has
been implicated in amyloid precursor protein processing. Astrocytes produce
significant levels of HtrA1 and Aß and application of an HtrA1 inhibitor leads to the
accumulation of Aß in cell culture supernatants. ß-secretase cleaves amyloid
precursor protein (APP) before Asp672 producing APP(672-770) which is further
cleaved by γ-secretase resulting in APP(672-711/713) referred as Aß, and a Cterminal fragment. To provide quality assurance for the investigation of the proteolytic
specificity of HtrA1, the primary structure of the recombinant APP(672-770) was
characterised by a combination of 1D-gel electrophoresis and mass spectrometry.
Proteolytic digestion by HtrA1 was analysed for APP(672-770) in comparison to
APP(672-711), APP(672-713), APP(724-770) and APP(661-687), and digestion
products were identified directly by high-resolution MALDI-FT-ICR. Digestion of
APP(672-770) was established to occur after residues Val-683, Gln-686, Asn-755,
and Asp-672 providing degradation products of approximately equal sequence
lengths. A consistent molecular fragmentation pattern for the C-terminal end of C99
was found when using APP(724-770) and Aß as substrates. However, additional
cleavage sites were observed at similar sequence lengths ranging between 10-20
residues.
The last part of the thesis was focused on mass spectrometric epitope elucidation of
a specific antibody to the H1-carbohydrate recognition domain. ESI-FT-ICR mass
spectrum of the intact H1CRD (17 kDa) and LC-MS of antigen tryptic mixtures
provided information on the structure of the antigen. The Met-1 residue was found to
be missing. A comparison between the MALDI-FT-ICR mass spectra of the tryptic
mixture of the antigen in native and reduced/alkylated form led to the identification of
2 disulfide bridges. The antigen was found to bind to the immobilised antibody in
Summary 134
both native and alkylated form. Epitope excision and extraction using trypsin led to
the identification of 2 N-terminal epitope peptides which exhibit affinity to the antibody.
Zusammenfassung 135
5. ZUSAMMENFASSUNG
Jüngste Fortschritte in der Immunologie und der Molekularbiologie haben zur
Entwicklung therapeutischer Impfstoffe geführt, die möglicherweise bei chronischen
Krankheiten wie Krebs, Herz-Kreislauf-Erkrankungen und neurodegenerativen
Krankheiten eingesetzt werden können, wo andere Therapien bisher kaum wirksam
sind. Dabei werden künftige Fortschritte in der Impfstoff-Entwicklung wesentlich von
einem besseren Verständnis der strukturellen Basis von Immunreaktionen abhängen.
Die Massenspektrometrie hat sich in den letzten Jahren zu einer weit verbreiteten
Methode zur Untersuchung von Struktur und Funktion von Proteinen sowie deren
Wechselwirkung mit anderen Biomolekülen entwickelt. Hauptmerkmale der
massenspektrometrischen Proteinanalyse sind hohe Empfindlichkeit und
Massengenauigkeit, kurze Analysenzeiten und geringer Substanzbedarf. Zur Analyse
komplexer Proteingemische und Strukturaufklärung molekularer
Erkennungsdomänen wurden zahlreiche Methoden in Kombination mit der
Massenspektrometrie entwickelt. Hierzu gehören chromatographische und
elektrophoretische Trennmethoden, proteolytische Assays, spezifische chemische
Modifizierung von Aminosäuren, Probenvorbereitungs- und Bioinformatik- Verfahren
zur Datenanalyse.
Ein Hauptmerkmal der Alzheimerschen Krankheit ist die Akkumulierung
extrazellulärer Plaques, die Aggregate des neurotoxischen ß-Amyloid- Peptids
enthalten, im menschlichen Gehirn. Immuntherapeutische Verfahren zur
Disaggregation der Plaques und/oder Inhibierung der Aß-Aggregation haben in den
letzten Jahren zunehmendes Interesse gefunden. Die Hauptzielsetzungen der ersten
beiden Teile der vorliegenden Dissertation lagen in der Entwicklung und Anwendung
massenspektrometrischer und immunanalytischer Methoden zur Identifizierung der
Epitope des Aß-Polypeptids, die von anti-Aß- Antikörpern erkannt werden.
Im ersten Abschnitt der Dissertation wurde die Wechselwirkung eines N-terminalen
Aß-(4-10)-Peptidepitops (FRHDSGY) mit mono- und polyklonalen Antikörpern
untersucht, das in früheren Arbeiten als strukturelles Epitop zweier Antikörper
identifiziert worden war (polyklonaler anti-Aß(1-42)- und monoklonaler anti-Aß(1-17)Zusammenfassung 136
Antikörper). Zur Charakterisierung der funktionelle Bedeutung der einzelnen
Aminosäuren wrden spezifische Mutagenese mit synthetischen Aß-(4-10) Mutanten
durchgeführt. Die Affinitätsbestimmung der Aß-Peptidmutanten erfolgte durch
vergleichende ELISA- Bindungsstudien. Bei der Wechselwirkung des polyklonalen
Antikörpers mit den Peptid-Mutanten D7A, S8A, G8A und Y10A wurden die
Aminosäuren F4, R5 und H6 als essentiell identifiziert, während im Fall des
monoklonalen Antikörpers alle Aminosäuren essentiell für die Bindung waren. Die
Bindung des Aß(1-16)- Peptids an zwei monoklonale Antikörper (Anti(1-17)-Klone
6E10 und Anti(1-40) Klone Bam-10) wurde zusätzlich in Gegenwart von Zn2+-Ionen
untersucht, die aufgrund von früheren massenspektrometrischen Untersuchungen
Wechslewirkung mit den His6-, His13- und His14- Resten zeigen. Für diese
Untersuchungen wurden die Peptide Aß(1-10) und Aß1-16) durch
Festphasenpeptidsynthese mittels Fmoc-Strategie mit einem zusätzlichen Spacer
aus fünf Glycinresten sowie Biotin am N-terminalen Ende synthetisiert. Beide Peptide
zeigten konzentrations-abhängige Bindung an die Antikörper. Die Anwesenheit von
Zn2+ -Ionen führte zu einer 4- bzw. 10-fachen Erhöhung der Bindung von Biotin-G5Aß(1-16) an die beiden Antikörper, während kein Effekt von Zn2+ auf die Bindung von
Biotin-G5-Aß(1-10). Im egensatz hierzu zeigten Co2+ und Ni2+ keinen Effekt auf die
Bindung der N-terminalen Aß-Peptide. Die Anwesenheit von Cu2+-Ionen zeigte
keinen Einfluß auf die Erkennung von Biotin-G5-Aß(1-16) an den mAb 6E10, jedoch
eine erhöhte Bndung an den BAM-10- Antikörper. Diese Ergebnisse zeigen einen
durch Zink induzierten Konformationseffekt auf die N-terminale Domäne Aß(1-16)
des Aß- Peptids, der zu einer erhöhten Bindung des Aß(4-10) Epitops an anti-AßAntkörper führt.
Der Hauptteil der Dissertation konzentrierte sich auf die Identifizierung des Epitops,
das durch natürlich vorkommende humane Aß-Autoantikörper erkannt wird. Die AßAntikörper wurden durch Affinitätschromatographie aus kommerziell erhältlichen
humanem Immunglobulin sowie Serum von Alzheimerpatienten isoliert. Als
Affinitätsmatrix für die Antikörperisolierung wurde eine Mikrosäule durch
Immobilisierung von Cys-Aß(1-40) an eine Iodacetylmatrix hergestellt. Zur
massenspektrometrischen Epitopidentifizierung wurde eine Affinitätssäule aus den
gereinigten Antikörpern hergestellt. Durch selektive proteolytische Spaltung von
intaktem, affinitätsgebundenem Aß mit Trypsin und Endoprotease-V8 und
Zusammenfassung 137
nachfolgender massenspektrometrischer Analyse der Epitop- und ÜberstandsFraktionen wurde nachgewiesen, daß das Epitop innerhalb der Sequenz Aß(12-40)
lokalisiert ist. Das durch Epitop-Exzision erhaltene Ergebnis wurde durch EpitopExtraktion bestätigt. Die Anwendung der Protease Pronase ergab die Identifizierung
von Aß(21-37) als minimale Epitopsequenz. Vergleichende Bindungsstudien von
humanen anti-Aß Antikörpern mit Aß(1-16), Aß(1-40), Aß(12-40) und Aß(17-28),
jeweils mit einem Spacer aus fünf Glycinresten sowie Biotin am N-terminalen
Sequenzende, wurden durch ELISA durchgeführt. Die Ergebnisse zeigten, daß Aß(116) und Aß(17-28) nicht an die Antikörpern binden, während Aß(12-40) und Aß(1-40)
konzentrationsabhängige Affinität aufweisen. Analoge Ergebnisse und identische
Epitoperkennung wurden für anti-Aß-Autoantikörper von Alzheimerpatienten
nachgewiesen. Durch Auftrennung mittels 2D-Gelelektrophorese konnte die
Polyklonalität der Antikörper charakterisiert werden. Massenspektrometrische
Untersuchungen der tryptischen Abbaugemische der Proteinbanden mittels MALDIFT-ICR-MS führten zur Identifizierung der schweren Antikörperketten vom γ1- und γ2Typ, sowie der leichten Ketten vom κ-Typ.
Ein weiterer Abschnitt der Doktorarbeit befaßte sich mit dem Effekt der Serinprotease
HtrA1 auf die Prozessierung des Amyloid- Vorläuferproteins. Astrozyten produzieren
bedeutende Mengen an HtrA1 und Aß, und die Zugabe eines HtrA1-Inhibitors führt
zu einer Akkumulierung von Aß in Zellkulturen. Die ß-Secretase spaltet das AmyloidVorläuferprotein (APP) vor Asp672 unter Bildung von APP(672-770), das in einem
zweiten Schritt durch die γ-Sekretase zu APP(672-711/713) (Aß) und einem Cterminalen Fragment gespalten wird. Zur Untersuchung der proteolytischen Spezifität
der HtrA1- Protease wurde zunächst die Primärstruktur von rekombinantem
APP(672-77) durch 1D-Gelelektrophorese, proteolytische Spaltung mit Trypsin und
Massenspektrometrie charakterisiert. Der proteolytische Abbau durch HtrA1 wurde
für APP(672-770) im Vergleich zu APP(672-711), APP(672-713), APP(724-770) und
APP(661-687) analysiert und die Abbauprodukte durch hochauflösende MALDI-FTICR-MS identifiziert. Die Spaltung von APP(672-770) wurde an den Aminosäuren
Val-683, Gln-686, Asn-755 und Asp-672 nachgewisen, jeweils unter Bildung von
Abbauprodukten von etwa gleicher Sequenzlänge. Es konnte ein molekular
einheitliches Fragmentierungsschema für die C-terminale (cytosolische) Domäne
nachgewiesen werden, das keine Priorität für spezifische Aminosäurereste besitzt,
Zusammenfassung 138
aber Produkte mit vergleichbaren Sequenzlängen von etwa 10-20 Aminosäuren
bildet.
Der letzte Abschnitt der Dissertation befaßte sich mit der massenspektrometrischen
Epitopbestimmung eines spezifischen Antikörpers gegen die H1-KohlenhydratErkennungsdomäne H1CRD. ESI-FTICR- Massenspektren des intakten H1CRDProteins, sowie ESI-LCMS- Fragmentierungsspektren von tryptischen
Abbaugemischen des Antigens lieferten Information über die Struktur des Antigens;
dabei wurde das Fehlen des Met-1 Rests nachgewiesen. Der Vergleich von MALDIFT-ICR- Massenspektren von tryptischen Gemischen des nativen, sowie des
reduzierten und alkylierten Antigens führte zur Identifizierung von zwei
Disulfidbrücken. Das Antigen zeigte Wechselwirkung zum immobilisierten Antikörper
in der nativen sowie der alkylierten Form. Durch Epitop-Exzision und –Extraktion mit
Trypsin konnten zwei N-terminale Epitop-Peptide identifiziert werden, die Affinität
zum Antikörper zeigen.
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Appendix 156
7. APPENDIX
7.1. Appendix 1
Abbreviations
Aß ß-amyloid peptide
Ab Antibody
ACN Acetonitrile
AD Alzheimer´s Disease
APP Amyloid precursor protein
APS Ammonium persulfate
ASGPR Asialo-glycoprotein receptor
BSA Bovine serum albumin
CDR Complementarity determining domain
CRD Carbohydrate recognition domain
Da Dalton
2-DE 2-dimensional gel electrophoresis
DHB 2,5-dihydroxybenzoic acid
m Mass difference
DMF N,N-Dimethylformamide
DTT 1,4-DL-Dithiothreitol
ELISA Enzyme linked immunosorbent assay
E:S Enzyme:Substrate ratio
ESI Electrospray ionisation
FT-ICR Fourier transform-ion cyclotron resonance
Fmoc 9-Fluorenylmethoxycarbonyl
HCCA Α-cyano-4-hydroxy-cynamic acid
HPLC High performance liquid chromatography
I.D. Internal dimensions ?
IEF Isoelectric focusing
IgG Immunoglobulin gamma
Appendix 157
IgM Immunoglobulin M
IPG Immobilised pH gradient
mAb Monoclonal antibody
MALDI Matrix-assisted laser desorption/ionisation
min minute
MS Mass spectrometry
m/z Mass-to-charge ratio
pAb Polyclonal antibody
PBS Phosphate buffer saline
pH Negative logarithmus of H3O
+ ion concentration
ppm Part per million
PyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium-PF6
- salt
RP Reversed phase
SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
T Tesla
TBS Tris buffer saline
TCA Trichloracetic acid
TEMED N,N,N´,N´-tetramethylethylenediamine
TFA Trifluoroacetic acid
TFE Trifluoroethanol
Tg Transgenic
TOF Time-of-flight
V Volt
ºC Grad Celsius
Xxx, Yyy Unspecified amino acid
Appendix 158
7.2. Appendix 2
Amino Acid 3/1 letter code
functional
group
Side chain
protecting group
MW
of the side chain
protecting group
Glycine Gly/G - - Alanine Ala/A - - Valine Val/V - - Leucine Leu/L - - Isoleucine Ile/I - - Proline Pro/P - - Aspartic
acid
Asp/D
-COOH 73
Glutamic
acid
Glu/E
-COOH
Serine Ser/S -OH
tert-butyl (tBu) 57.1
Threonine Thr/T -OH
Tyrosine
Tyr/Y -OH
Histidine His/H
trityl (Trt)
243.3
Cysteine Cys/C -SH
Glutamine Gln(Q) -CONH2
Asparagine Asn/N -CONH2
Arginine
Arg/A
2,2,4,6,7-pentamethyldihydrobenzofurane-6sulfonyl
253.3
Cysteine
-SH
72.1
Lysine
Lys/K
NH2
101.1
Tryptophane Trp/W
CH3
CH3
CH3
O
CH3
CH3
CH3
CH2 NH C CH3
O
OS
O
O
CH3
CH3
CH3
CH3CH3
NN Hτ
2
NH C NH
NH
OC
O CH3
CH3
CH3
Appendix 159
7.3. Appendix 3
Figure 83: MALDI-TOF mass spectra of the a) supernatant, b) wash and c) elution fractions of the
epitope extraction using GluC. The results indicate that the shorter N-terminal and midddomain sequences have no affinity to the Aß-autoantibody whereas the fragment
containing the middle and the carboxy-terminal part bind to the antibody.
Aß(1-40)
Aß(12-22)
Aß(12-40)
Aß(4-40)
Aß(4-11)
a) b) Aß(1-40)
Aß(12-40)
Aß(4-40)
Aß(1-40)
c) Appendix 160
Aß(1-40)
Aß(1-40)
Aß(1-40)
Aß(12-40)
Aß(4-40)
Aß(1-40)
Aß(12-22)
d) c) b) a) Figure 84: MALDI-TOF mass spectra of the a) Aß binding fraction b) supernatant, c) wash and d)
elution fractions of the epitope excision using GluC.
Appendix 161
Aß(12-22)
Aß(12-40)
Aß(4-11)
a) b) c) Figure 85: MALDI-TOF mass spectra of the epitope extraction using the affinity media containing nonspecific IgG: a) supernatant, b) washing and c) elution fraction
Appendix 162
Figure 86: MALDI-TOF mass spectra of the epitope excision using the affinity media containing nonspecific IgG: a) binding, b) supernatant, c) washing and d) elution fraction.
Aß(1-40)
[M+2H]2+
Aß(1-40)
Aß(1-40)
d) c) b) b) Appendix 163
7.4. Appendix 4
a) b) Edman, N-terminal, MALDI-TOF-MS, MALDI-FTMS, MS / MS = CDRs
1 EVQLVESGGG VVQPGGSLRL SCAASGFTFR SYWMSWVRQA PGKGLEWVAS
51 VKQDGSEKYY VDSVKGRFTI SRDTSKNTLY LQMNSLRAED TAVYYCARDA
101 SSWYRDWFDP WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV
151 KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ
201 TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK
251 PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY
301 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP
351 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP
401 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG
451 K
Interchain disulfide bridge Intrachain disulfide bridge 2D-SDS-PAGE/MALDI-FTMS
1 EIVLTQSPAT LSLSPGERVT ITCRESQGIR NYLAWYQQKP GQAPRLLIYG
51 ASTRATGIPD RFSGSGSGTD FTLTISRLEP EDFAVYYCQQ YGSSQGTFGP
101 GTKVDIKR T VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK
151 VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ
201 GLSSPVTKSF NRGEC
Edman, N-terminal, MALDI-TOF-MS, MALDI-FTMS, MS / MS = CDRs
Interchain disulfide bridge Intrachain disulfide bridge 2D-SDS-PAGE/MALDI-FTMS

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