A practical approach to enrich intact tryptic N-glycopeptides through size exclusion chromatography and hydrophilicity (SELIC) using an acrylamide-agarose composite gel system.

Increasing researches proved that abnormal glycosylation is strongly correlated with many diseases. Specially, site-specific glycosylation and its associated heterogeneity are closely related to the function and activity of the glycoprotein. However, intact N-glycopeptide analysis still faces great challenges because the presence of highly abundant non-glycosylated peptides would suppress the ionization of lowly abundant glycopeptides. In the present study, we developed a practical intact tryptic N-glycopeptide enrichment method using acrylamide-agarose composite gel that combined the size exclusion chromatography and hydrophilic (named SELIC) effects, aimed to remove the detergent rapidly and effectively, as well as enrich intact N-glycopeptides while extracting peptides. This is a useful tool to facilitate the intact N-glycopeptides analysis of complex protein mixtures, particularly for samples that extracted from formalin-fixed and paraffin-embedded (FFPE) tissues by SDS. Using this method, we successfully identified 700 site-specific intact tryptic N-glycopeptides corresponding to 261 glycosylation sites on 191 glycoproteins from FFPE thymoma tissues.