title: Carbonyl-reactive tandem mass tags for the proteome-wide quantification of N-linked glycans.
abstract: N-Linked protein glycosylation is one of the most prevalent post-translational modifications and is involved in essential cellular functions such as cell-cell interactions and cellular recognition as well as in chronic diseases. In this study, we explored stable isotope labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel approach for the quantification of N-linked glycans. Glyco-TMTs bearing hydrazide- and aminooxy-functionalized groups were compared for glycan reducing end derivatization efficiency and quantification merits. Aminooxy TMTs outperform the hydrazide reagents in terms of labeling efficiency (>95% vs 65% at 0.1 μM) and mass spectrometry based quantification using heavy/light-TMT labeled glycans enabled accurate quantification in MS1 spectra (CV < 15%) over a broad dynamic range (up to 1:40). In contrast, isobaric TMT labeling with quantification of reporter ions in tandem mass spectra suffered from severe ratio compression already at low sample ratios. To demonstrate the practical utility of the developed approach, we characterized the global N-linked glycosylation profiles of the isogenic human colon carcinoma cell lines SW480 (primary tumor) and SW620 (metastatic tumor). The data revealed significant down-regulation of high-mannose glycans in the metastatic cell line.
authors.affiliation: Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Hahne
authors.forename: Patrick
authors.initials: P
authors.lastname: Neubert
authors.forename: Karsten
authors.initials: K
authors.lastname: Kuhn
authors.forename: Chris
authors.initials: C
authors.lastname: Etienne
authors.forename: Ryan
authors.initials: R
authors.lastname: Bomgarden
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D050050:Protein Carbonylation; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D020543:Proteome