Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation
Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30% to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE valuespreferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thus enabling the putative glycan structure characterization. Also, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. On the contrary, HCD with higher NCE values preferentially fragmented the peptide backbone and thus provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N-linked and O-linked glycopeptides in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (including six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both, the peptide and the glycan, thus enabling comprehensive structural characterization of glycoproteins in a single LC-MS/MS analysis.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1129287
- Report Number(s):
- PNNL-SA-99189; 45394; KP1704020
- Journal Information:
- Analytical Biochemistry, 452:96-102, Journal Name: Analytical Biochemistry, 452:96-102
- Country of Publication:
- United States
- Language:
- English
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