N-Glycopeptide Reduction with Exoglycosidases Enables Accurate Characterization of Site-Specific N-Glycosylation.
We report a new approach called NGlycoReduction that generates an oligomannosylated N-glycopeptidome by enzymatically removing sugars outside the N-glycopeptide pentasaccharide core with exoglycosidases. This approach is based on our discovery that the fragmentation of glycopeptides is glycan-structure dependent and glycans with core mannose structures overwhelmingly lead to the generation of Y1 ions when subjected to MS/MS in mass spectrometry. Oligomannosylated glycopeptidome produced by NGlycoReduction can be mixed with the intact N-glycopeptidome and analyzed by HPLC-ESI-MS together to enable the identification of peptide sequence, glycosylation site and the structure of intact glycopeptides. The glycan structure of intact glycopeptides can be identified from MS/MS spectra of their own and their peptide sequences were identified by the MS3spectra of the oligomannosylated glycopeptides with the same Y1 ion. Both mass tolerance and difference in retention time were further used to increase the confidence in the Y1 ion alignment. This approach has the advantage of low cost and ease of processing and can be expanded to other samples, especially for characterizing site-specific N-glycosylation involving complex N-glycans. In this study, simultaneous analysis of the combined oligomannosylated N-glycopeptidome and the native glycopeptidome leads to the identification of 609 N-glycopeptides from the secretome and lysates of Huh7 cells.
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Rui
R
Chen
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Kai
K
Cheng
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Zhibin
Z
Ning
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Daniel
D
Figeys
D016000:Cluster Analysis; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014407:Tumor Cells, Cultured
D006020:Glycopeptides; D011134:Polysaccharides; D006026:Glycoside Hydrolases