Journal
JOURNAL OF PROTEOME RESEARCH
Volume 16, Issue 4, Pages 1706-1718Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.6b01053
Keywords
glycoprotein; glycoconjugate; glycoproteomics; chemical biology; LC-MS/MS; metabolic labeling chemical enrichment; glycomics; IsoStamp
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Funding
- US National Institutes of Health [CA200423, U01 CA207702, P41GM10349010, 1S10OD018530, 1S10OD020062-01]
- Jane Coffin Childs Memorial Fund
- Burroughs Wellcome Fund Career Awards at the Scientific Interface
- Stanford Undergraduate Advising and Research Student Grant
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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 Ac(4)GalNAz or Ac(4)ManNAz 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.
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