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. 2011 Oct;46(10):993-1003.
doi: 10.1002/jms.1980.

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

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Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

Farid Jahouh et al. J Mass Spectrom. 2011 Oct.

Abstract

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.

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Figures

Figure 1
Figure 1
Schematic representation of the general structure of carbohydrate–protein constructs from oligosaccharide fragments of the antigenic tetrasaccharide side chain of the Bacillus anthracis exosporium.
Figure 2
Figure 2
General strategy applied for the mass spectrometry determination of the glycation sites on the hapten–BSA glycoconjugate.
Figure 3
Figure 3
(a) MALDI-MS analysis of the tryptic carbohydrate hapten–BSA glycoconjugate digests. (b) MALDI-MS analysis of the digested carbohydrate hapten–BSA glycoconjugate with the GluC V8 endoproteinase.
Figure 4
Figure 4
(a) MALDI-TOF/TOF-MS/MS spectra of the glycated peptide ALK*AWSVAR (Lys 235) at m/z 1951.0130. (b) Different product ions involving the fragmentation of the carbohydrate hapten observed during the MALDI-TOF/TOFMS/MS analysis of the glycated peptide ALK*AWSVAR (Lys 235) at m/z 1951.0130.
Figure 5
Figure 5
LC-QqTOF-MS/MS spectra of the tryptic glycated peptides (a) CCTK*PESER (Lys 463) at m/z 1058.4652 (+2), (b) CASIQK*FGER (Lys 228) at m/z 1073.0144 (+2) and GluC V8 digests, (c) K*VTKCCTE (Lys 495) at m/z 987.9658 (+2) and (d) K*QEPERNE (Lys 117) at m/z 989.9697 (+2).
Figure 6
Figure 6
(a) BSA sequence where the glycation sites are indicated by an asterix (red = identified on tryptic digests, blue = identified on GluC V8 digests and red and underlined = identified on both tryptic and GluC V8 digests) and (b) 3D-structure of the BSA. The glycated lysine residues are highlighted in red (Swiss-Pdb Viewer software).

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