Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC–MS/MS

Abstract Glycosylation of membrane proteins plays an important role in cellular behaviors such as cell–cell interaction, immunologic recognition and cell signaling. However, the effective extraction of membrane proteins, the selective isolation of glycopeptides and the mass spectrometric characterization of glycosylation are challenging with current analytical techniques. In this study, a systematic approach was developed which combined: an integrated hydrophilic interaction chromatography solid phase interaction (HILIC SPE) for simultaneous detergent removal and glycopeptide enrichment, and mass spectrometric identification of both protein N-glycosylation sites and site-specific glycan composition. The HILIC SPE conditions were optimized to enable the use of a high concentration of strong detergents, such as SDS and Triton X-100 and to dissolve highly hydrophobic membrane proteins, thus increasing the yield of membrane protein extraction. We illustrated the performance of this approach for the study of membrane protein glycosylation from human embryonic kidney cell lines (HEK 293T). 200 μg total protein digest was processed using this approach, leading to the identification of 811 N-glycosylation sites from 567 proteins within two experimental replicates. Furthermore, 177 glycopeptides representing 82 N-glycosites with both glycan composition and peptide sequence were identified by high energy collision dissociation. Biological significance A method for systematic characterizing of cell membrane glycosylation has been developed in this manuscript. It is comprised of an integrated hydrophilic interaction chromatography solid phase extraction for the simultaneous detergent removal and intact glycopeptide enrichment. This HILIC SPE significantly increased the efficiency and sensitivity for glycosylation analysis and was combined with high energy collision dissociation to characterize site-specific N-glycosylation from HEK293 cell membrane. Totally 811 N-glycosylation sites from 567 proteins were identified and 177 intact glycopeptides with both glycan composition and peptides sequence were characterized, which provided a solution for site-specific N-glycosylation characterization of membrane.