2. RESULTS AND DISCUSSION
2.2. Epitope elucidation of Aß-specific human antibodies
2.2.4. Clonal diversity and sequence analysis of human serum Aß
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
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
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 Aß-autoantibody 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 anti-lysozyme antibody visualized by silver staining.
6 7 8 9
4 5
pI pI 4 5 6 7 8 9
Heavy chain
Light chain
Heavy chain
Light chain
6 7 8 9
4 5
pI 4 5 6 7 8 9 pI 4 5 6 7 8 9
pI pI 4 5 6 7 8 9
Heavy chain
Light chain
Heavy chain
Light chain
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.
[96-106][372-382]
[149-160][388-397] [316-328] [302-315] [329-344] [283-301] [398-419] [444-466] [250-275] [246-275]
[96-106][372-382]
[149-160][388-397] [316-328] [302-315] [329-344] [283-301] [398-419] [444-466] [250-275] [246-275]
Figure 37: Mass spectrum of the peptide mixture resulted by trypsin treatment of the spot 12.
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 [101-105] comprised by the tryptic sequence [95-[101-105] are part of the CDR.
In the case of the protein spots annotated for the light chain molecules of the Aß-autoantibodies (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.
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
[62-77] [108-126] [109-126] [127-142] [150-169] [189-207] [191-207]
[62-77] [108-126] [109-126] [127-142] [150-169] [189-207] [191-207]
Figure 40: Mass spectrum of the peptide mixture resulted by trypsin treatment of the spot 4.
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.
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 [23-40]. 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)
Aß(4-11) Aß(2-11)
Aß(1-11)
Aß(23-40)
Aß(12-40)
Aß(4-40) Aß(1-40) 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)
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] 23
VGSNKGAIIGLMVGGVV40 3020.6561 3020.6503 2 [12-40] 12
VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40 3036.6266 3036.6497 7 [12-40]* 12
VHHQKLVFFAEDVGSNKGAIIGLM*VGGVV40 4012.0549 4012.0651 3 [4-40] 4
FRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40 4327.0248 4327.1717 49 [1-40] 1DAEFRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVV40
* Oxidation at Met-35
2.2.6. Synthesis of biotinylated amyloid peptides encompassing the