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

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.

蛋白质糖基化可产生丰富的生物学后果，这些后果反映了糖链结构中所编码的分子多样性。这种相同的结构多样性对研究完整的糖蛋白组方法的发展提出了重大挑战。我们最近引入了一种称为同位素靶向糖蛋白组学（IsoTaG）的方法，该方法利用同位素重编码来表征带有完整糖链的叠氮糖标记糖肽。在此，我们描述了该方法在分析来自一系列组织多样细胞系的糖蛋白组中的广泛应用。这一努力得益于一种名为IsoStamp v2.0 的新型高保真模式搜索和糖肽验证算法，以及新型的稳定同位素探针。将IsoTaG平台应用于用Ac4GalNAz或Ac4ManNAz代谢标记的15种细胞线，揭示了1375种N-糖肽和2159种O-糖肽，它们以74种不同的糖链结构进行修饰。IsoTaG观察到的糖肽结合的糖链与通过甲硫醇化分析确定的释放的N-糖链相媲美。因此，IsoTaG定位用于增强对糖蛋白组结构的结构理解。