Title : 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)
- 200μg 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