Figure 36.

General scheme of bottom-up glycoproteomics workflows. Glycoproteins are extracted and enriched from biological samples. The glycoproteins are digested by proteolytic enzymes, and the resulting glycopeptides are enriched from the digestion mixture. The concentrated glycopeptides are separated via HPLC or CE to facilitate isomeric identification and subsequently analyzed via MS/MS. Complete glycopeptide characterization requires three elements: the sequencing of the peptide backbone, the sequencing of the glycan moiety, and the localization of the glycosylation site within the amino acid sequence.