Highly efficient N-glycoproteomic sample preparation by combining C-18 and graphitized carbon adsorbents

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 N-glycopeptides 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 N-glycopeptides with 1,759 unique N-glycosylation sites were finally identified