Highly efficient N-glycoproteomic sample preparation by combining C_(18) and graphitized carbon adsorbents

摘要

Conventional N-glycoproteome analysis usually applies C_(18) reversed-phase (RP) adsorbent for sample purification, which will lead to unavoidable sample loss due to the high hydrophilicity of N-glycopeptides. In this study, a porous graphitized carbon (PGC) absorbent was combined with a C_(18) adsorbent for N-glycopeptide purification in comprehensive N-glycoproteome analysis based on the hydrophobic and polar interactions between carbon and N-glycans. It was observed that the small hydrophilic N-glycopeptides that cannot retain onto C_(18) adsorbent can be captured by the graphitized carbon, while the large hydrophobic N-glycopeptides that cannot retain onto the graphitized carbon can be feasibly captured by the C_(18) adsorbent. Comparing with sample purification by using C_(18) adsorbent only, 28.5 more Nglycopeptides were identified by combining both C_(18) and PGC adsorbents. The C_(18)-PGC strategy was further applied for both sample purification and pre-fractionation of a complex protein sample from HeLa cell. After hydrophilic interaction chromatography enrichment, 1,484 unique Nglycopeptides with 1,759 unique N-glycosylation sites were finally identified.