Woo Christina M, Felix Alejandra, Byrd William E, Zuegel Devon K, Ishihara Mayumi, Azadi Parastoo, Iavarone Anthony T, Pitteri Sharon J, Bertozzi Carolyn R
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.
J Proteome Res. 2017 Apr 7;16(4):1706-1718. doi: 10.1021/acs.jproteome.6b01053. Epub 2017 Feb 28.
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 AcGalNAz or AcManNAz 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各种各样的生物学后果，这反映了聚糖结构中编码的分子多样性。同样的结构多样性给完整糖蛋白质组研究方法的开发带来了重大挑战。我们最近引入了一种称为同位素靶向糖蛋白质组学（IsoTaG）的方法，该方法利用同位素重新编码来表征带有完全完整聚糖的叠氮糖标记糖肽。在此，我们描述了该方法在分析来自多种组织的细胞系糖蛋白质组方面的广泛应用。这项工作得益于一种名为IsoStamp v2.0的新型高保真模式搜索和糖肽验证算法以及新型稳定同位素探针。将IsoTaG平台应用于用AcGalNAz或AcManNAz进行代谢标记的15个细胞系，共鉴定出1375个N-糖肽和2159个O-糖肽，这些糖肽带有74种不同的聚糖结构修饰。通过IsoTaG观察到的糖肽结合聚糖与通过甲基化分析鉴定出的释放型N-聚糖相当。因此，IsoTaG有助于增强对糖蛋白质组的结构理解。
