Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation.

摘要: Simultaneous elucidation of the glycan structure and glycosylation site are needed to reveal biological function protein glycosylation. In this study, we employed a recent type fragmentation termed higher energy collisional dissociation (HCD) examine patterns intact glycopeptides generated from mixture standard glycosylated proteins. The normalized (NCE) value for HCD was varied 30 60% evaluate optimal conditions peptide backbones glycoconjugates. Our results indicated that with lower NCE values preferentially fragmented sugar chains attached peptides generate ladder neutral loss monosaccharides, thereby enabling putative characterization. addition, detection oxonium ions enabled unambiguous differentiation non-glycopeptides. contrast, backbone and, thus, provided information confident identification. We evaluated approach alternating parameters characterization N- O-linked in single liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. applied novel data analysis pipeline, so-called GlycoFinder, form basis automated Overall, 38 unique corresponding eight sites (six N-linked two sites) were confidently identified mixture. This concurrent both glycan, comprehensive structural glycoproteins LC-MS/MS