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J Proteome Res. 2017 Apr 7;16(4):1706-1718. doi: 10.1021/acs.jproteome.6b01053. Epub 2017 Feb 28.

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes.

Woo CM1,2,3,4, Felix A1,2,3,4, Byrd WE1,2,3,4, Zuegel DK1,2,3,4, Ishihara M1,2,3,4, Azadi P1,2,3,4, Iavarone AT1,2,3,4, Pitteri SJ1,2,3,4, Bertozzi CR1,2,3,4.

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Department of Chemistry; §School of Engineering; #Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine; ∇Howard Hughes Medical Institute, Stanford University , Stanford, California 94305, United States.
School of Computing, University of Utah , Salt Lake City, Utah 84112, United States.
Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
QB3Mass Spectrometry, University of California , Berkeley, California 94720, United States.


Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by IsoTaG were found to be comparable to released N-glycans identified by permethylation analysis. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.


IsoStamp; LC−MS/MS; chemical biology; chemical enrichment; glycoconjugate; glycomics; glycoprotein; glycoproteomics; metabolic labeling

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