Glycosylation is a common protein PTM, and its aberrant regulation has been widely linked to various pathological conditions including cancers. Our recent development of intact N-glycopeptide search engine GPSeeker has enabled relatively quantitative structure-specific characterization of differentially expressed intact N-glycopeptides (DEGPs) with isotopic labeling of the peptide backbones and structure-specific fragment ions of the N-glycan moieties. Here we report our site- and structure-specific relatively quantitative N-glycoproteomics study of breast MCF-7 cancer cells (relative to epithelial MCF-10A cells) using RPLC-nanoESI-MS/MS and GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones (amino acid sequences, N-glycosites) and the N-glycan moieties (monosaccharide composition, sequence and linkages) were identified from five technical replicates (TR1, TR2, TR3, TR4 and TR5). With the criteria of quantified at least thrice out of the five technical replicates with no <1.5-fold change, p ≤ .05 and RSD ≤ 20%, 56 DEGPs were quantified from 23 N-glycosites on 19 intact N-glycoproteins. For the 19 intact N-glycoproteins observed with differential N-glycosylation expression, 14 (each with one or more DEGPs) were observed with uniform down regulation; 6 (each with one or more DEGPs) were observed with uniform up regulation; whereas one was observed with both up and down regulation. SIGNIFICANCE: Differential N-glycosylation in breast MCF-7 cancer cells (relative to MCF-10A epithelial cells) were qualitatively and quantitatively characterized with site- and structure-specific N-glycoproteomics using RPLC-nanoESI-MS/MS (HCD with stepped NCEs) and intact N-glycopeptide search engine GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones and the N-glycan moieties were identified; For the 248 putative N-glycosites, 248 were confirmed where 125 have not been annotated in UniProt as of July 25, 2019. For the 114 N-glycan putative linkage structures, 44 were confirmed with no less than one structure-diagnostic fragment ions. With the criteria of quantified at least thrice out of the five technical replicates with no < 1.5-fold change and p ≤ .05, 56 DEGPs were quantified from 21 intact N-glycoproteins; 13 and 5 intact N-glycoproteins (each with one or more DEGPs) were observed with uniform down and up regulation; whereas one were observed with simultaneous up and down regulation.

中文翻译：

---

特定于位点和结构的定量N-糖代谢组学研究MCF-7癌细胞中N-糖基化的差异。

糖基化是一种常见的蛋白质PTM，其异常调节已广泛与包括癌症在内的各种病理状况联系在一起。我们对完整N-糖肽搜索引擎GPSeeker的最新开发已经实现了差异表达的完整N-糖肽（DEGP）的相对定量结构特异性表征，包括肽主链的同位素标记和N-聚糖部分的结构特异性片段离子。在这里，我们报告使用RPLC-nanoESI-MS / MS和GPSeeker对乳腺MCF-7癌细胞（相对于上皮MCF-10A细胞）的特定于位点和结构的相对定量N-糖代谢组学研究。光谱水平FDR≤1％，完整的581个N-糖肽具有两个肽主链的全面结构信息（氨基酸序列，从五个技术重复样本（TR1，TR2，TR3，TR4和TR5）中鉴定了N-糖基）和N-聚糖部分（单糖组成，序列和键）。采用五个技术重复样本中至少有三次的定量标准，且没有<1.5倍变化，p≤.05和RSD≤20％，从19种完整的N-糖蛋白上的23种N-糖苷中定量了56种DEGP。对于观察到的19个完整的N-糖蛋白，它们具有不同的N-糖基化表达，观察到14个（均带有一个或多个DEGP），并且具有统一的下调作用。观察到6（每个都有一个或多个DEGP）具有统一的上调；而有人观察到向上和向下调节。意义：使用RPLC-nanoESI-MS / MS（HCD和阶梯状NCE）和RPLC-nanoESI-MS / MS通过定点和结构特异性N-糖代谢组学定性和定量表征乳腺癌MCF-7癌细胞（相对于MCF-10A上皮细胞）中的N-糖基化完整的N-糖肽搜索引擎GPSeeker。在光谱水平FDR≤1％的情况下，鉴定了581个完整的N-糖肽，具有完整的肽主链和N-糖基结构信息。截至2019年7月25日，对于248种假定的N-糖苷，已确认有248种在UniProt中尚未注释的125种。对于114种N-聚糖的假定键合结构，已确认44种具有至少一个结构诊断碎片离子。在五个技术重复样本中，至少有三次被量化，没有<1.5倍变化且p≤.05，从21种完整的N-糖蛋白中定量了56种DEGP；观察到13和5个完整的N-糖蛋白（每个都有一个或多个DEGPs）具有统一的上下调节；而观察到一个同时向上和向下调节。