2019 Volume 4 Issue 2 Pages 71-81
Protein glycosylation is one of widely occurred post-translational modifications. Methods for large-scale identification of glycosylated proteins were developed in the early days of field of proteomics. At that time, direct analysis of intact glycopeptide was difficult due to the structural factors. Major reasons of the difficulty were that glycan is diverse and heterogeneous, and glycopeptide is a conjugate composed of two oligomers (peptide and glycan) formed by different energy of bonds. Therefore, glycosylated peptides were identified after removal of glycan to specify glycosylation site. To ensure that the site was actually glycosylated, we used stable isotope labeled water (H218O) to label the site (IGOT method) during glycan removal by Peptide-N-glycanase (PNGase) action. Then, if sample glycopeptides are captured by the lectin affinity chromatography, glycan motif on the captured glycopeptides is presumable based on the lectin specificity. Using the lectin well reflecting disease-related glycan alteration, we have captured glycopeptides to identify glycoproteins as biomarker candidates. Currently, such glycoprotein possessing disease-specific glycan, especially membrane glycoprotein, are thought to be a potential pharmaceutical target, thus we developed a new high sensitivity technology to assign intact glycopeptides relying MS1 rather than MS2 (Glyco-RIDGE method). In this review, we introduce development and applications of glycoproteomic technologies we made.
