Open AccessIsotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes
Author(s) -
Woo Christina M.,
Bertozzi Carolyn R.
Publication year - 2016
Publication title -
current protocols in chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.503
H-Index - 14
ISSN - 2160-4762
DOI - 10.1002/9780470559277.ch150185
Subject(s) - glycoproteomics , glycosylation , proteome , chemistry , glycopeptide , glycan , biochemistry , mass spectrometry , proteomics , computational biology , glycoprotein , biology , chromatography , gene , antibiotics
Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post‐translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this protocol, we describe a novel mass‐independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). To use IsoTaG, cell culture samples are metabolically labeled with an azido‐ or alkynyl‐sugar. Metabolically labeled glycoproteins are then tagged using Click chemistry and enriched with an isotopic recoding biotin probe. Intact glycopeptides are recovered by cleavage of the probe, analyzed with directed MS, and assigned by targeted mass‐independent data analysis. The outlined procedure is well defined in cell culture and has been executed with over 15 cell lines. © 2016 by John Wiley & Sons, Inc.