Glycoproteomics methods and tools

Direct mass analysis of glycoprotein samples is very challenging because of their heterogeneity. Therefore, samples are digested with proteases (Endoproteinase AspN ,  Endoproteinase GluC, Endoproteinase LysC, Trypsin-ultra^™, Mass Spectrometry Grade) and glycopeptides can be enriched for subsequent analysis. However, even reduced to the peptide level, direct structural elucidation by LC-MS/MS is difficult compared with routine mapping of unmodified peptides.

• Fragmentation by CID results in glycan cleavages, leaving no diagnostic fragments for peptide sequencing. Instead, Electron Transfer Dissociation (ETD) is used with glycopeptides to obtain peptide fragments that allow more precise identification.
• Alternative proteolytic treatments are used to generate overlapping, identifying fragments.
• Combined glycosidase treatments allow the unequivocal identification of N- and O-glycan sites.
• Deglycosylation by endoglycosidases or amidases leads to identification of partially occupied N-glycan sites, which are otherwise predicted as N-glycan sequons.

References:

1. Muchena, J. et al. (2014) Anal. Chem. 86(1): 196 - 212. PMID 24313268