Comparison of Enrichment Methods for Intact N- and O‑Linked Glycopeptides Using Strong Anion Exchange and Hydrophilic Interaction Liquid Chromatography

Heterogeneity of protein glycosylation poses great challenges for analysis that is key to understand structure and function of glycoproteins. Resolving this conundrum requires efficient and specific enrichment of intact glycopeptides for identification and quantitation. To this end, hydrophilic interaction chromatography (HILIC) has been commonly used to enrich intact N- and O-linked glycopeptides. However, its effectiveness to enrich isobarically labeled glycopeptides remains unclear. Here, we studied three different enrichment methods for enrichment of N- and O-linked glycopeptides. It was found that removal of N-glycans prior to enrichment of O-linked glycopeptides by HILIC improved identification of O-linked glycopeptides by mass spectrometry. We also compared the enrichment of intact N- and O-linked glycopeptides using other chromatography methods and found that using cartridges containing materials for strong anion exchange (SAX) chromatography increased yield and identification of N- and O-linked glycopeptides. The enrichment of O-linked glycopeptides was further improved when a Retain AX cartridge (RAX) was used. In particular, isobaric tag labeled glycopeptides after C18 desalting could be readily enriched by SAX and RAX cartridges but not by HILIC to enable quantitative glycoproteomics. It is anticipated that the use of SAX and RAX cartridges will facilitate broad applications of identifications and quantitation of glycoproteins