TMT-Based Multiplexed Quantitation of N‑Glycopeptides Reveals Glycoproteome Remodeling Induced by Oncogenic Mutations

Glycoproteomics, or the simultaneous characterization of glycans and their attached peptides, is increasingly being employed to generate catalogs of glycopeptides on a large scale. Nevertheless, quantitative glycoproteomics remains challenging even though isobaric tagging reagents such as tandem mass tags (TMT) are routinely used for quantitative proteomics. Here, we present a workflow that combines the enrichment or fractionation of TMT-labeled glycopeptides with size-exclusion chromatography (SEC) for an in-depth and quantitative analysis of the glycoproteome. We applied this workflow to study the cellular glycoproteome of an isogenic mammary epithelial cell system that recapitulated oncogenic mutations in the PIK3CA gene, which codes for the phosphatidylinositol-3-kinase catalytic subunit. As compared to the parental cells, cells with mutations in exon 9 (E545K) or exon 20 (H1047R) of the PIK3CA gene exhibited site-specific glycosylation alterations in 464 of the 1999 glycopeptides quantified. Our strategy led to the discovery of site-specific glycosylation changes in PIK3CA mutant cells in several important receptors, including cell adhesion proteins such as integrin β-6 and CD166. This study demonstrates that the SEC-based enrichment of glycopeptides is a simple and robust method with minimal sample processing that can easily be coupled with TMT-labeling for the global quantitation of glycopeptides.

糖蛋白质组学（Glycoproteomics）指同时解析聚糖（glycans）及其所连接肽段（peptides）的技术，如今正愈发广泛地用于大规模构建糖肽（glycopeptides）图谱。然而，即便串联质量标签（TMT）这类等压标记试剂已常规应用于定量蛋白质组学，定量糖蛋白质组学仍颇具挑战。本研究提出一种工作流程，将TMT标记糖肽的富集或分级分离与尺寸排阻色谱（SEC）相结合，以实现糖蛋白质组（glycoproteome）的深度定量分析。我们将该工作流程应用于研究一种同基因乳腺上皮细胞系的细胞糖蛋白质组，该细胞系可模拟编码磷脂酰肌醇3-激酶催化亚基的PIK3CA基因的致癌突变。与亲本细胞相比，携带PIK3CA基因第9外显子（E545K）或第20外显子（H1047R）突变的细胞，在被定量的1999个糖肽中，有464个出现了位点特异性糖基化改变。本策略还发现，PIK3CA突变细胞中多个重要受体存在位点特异性糖基化变化，其中包括整合素β-6、CD166等细胞黏附蛋白。本研究证实，基于尺寸排阻色谱的糖肽富集法操作简便、稳定性强，且样品处理步骤极少，可轻松与TMT标记技术结合，实现糖肽的全局定量分析。