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

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.

Revealing the glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1, serotype Ogawa with the BSA protein carrier using LC‐ESI‐QqTOF‐MS/MS

“…However, the product at m/z 336.1918 was also detected in our previous work on Bacillus anthracis neoglycoconjugate vaccine model and assigned to the structure: [C 17 H 26 N 3 O 4 ] + which corresponds to a linker portion attached to a lysine residue (Fig. ) . Similarly, the product ion [C 11 H 16 N 3 O 2 ] + at m/z 222.1187 was produced from a linker portion attached to a lysine residue.…”

“…We recently reported the determination of the glycation sites in Bacillus anthracis neoglycoconjugate vaccine by MALDI‐TOF/TOF‐collision‐induced dissociation (CID)‐MS/MS and liquid chromatography (LC)‐ESI‐ quadrupole (Qq)TOF‐MS/MS . Among other things, it permitted us to evaluate the hapten‐to‐BSA ratio which was found to be 5.4:1.…”

Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C‐fragment carrier

“…In our previous studies, [21] we demonstrated the advantage of using LC-ESI-QqTOF-MS/MS, rather than MALDI-MS/MS, for establishing the glycation sites in glycoconjugates in which the carbohydrate portion is of low molecular weight (up to 950 Da). [21] Indeed, the LC-MS/MS analysis of the Bacillus anthracis neoglycoconjugate vaccine digests allowed us to identify 18 glycation sites, while only five glycation sites were observed during the MALDI-MS/MS analysis.…”

“…[21] Indeed, the LC-MS/MS analysis of the Bacillus anthracis neoglycoconjugate vaccine digests allowed us to identify 18 glycation sites, while only five glycation sites were observed during the MALDI-MS/MS analysis. [21] …”

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

“…Although there have been multiple reports showing the capabilities of using CID or ETD alone to sequence sugar-modified peptides, most often the data were collected only with a few protein standards that were modified by less complex glycation or O- glycosylation. It is not applicable to large-scale characterization of much more branched and complex glycosylated peptides, such as N- glycopeptides where glycan fragments dominate MS/MS spectra, and it is especially challenging to work with complex proteome samples [19–24]. An alternative approach for a more detailed characterization of glycopeptides combines MS/MS and MS 3 experiments with CID or HCD.…”