Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars
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Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.
KeywordsGlycoproteomics Chemical proteomics LC-MS/MS Metabolic labeling Sialic acid
Financial support from the US National Institutes of Health (CA200423, C.R.B.), Jane Coffin Childs Memorial Fund (C.M.W.), Burroughs Wellcome Fund Career Awards at the Scientific Interface (C.M.W.), Stanford Undergraduate Advising and Research Student Grant (A.F.), the W.M. Keck Foundation Medical Research Program (J.E.E.), and the Bill and Melinda Gates Foundation (J.E.E.) are gratefully acknowledged.
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Conflict of interest
The authors declare that they have no conflict of interest.
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