School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai 200092, China.
School of Chemical Science & Engineering and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai 200092, China.
J Proteomics. 2020 Feb 10;212:103594. doi: 10.1016/j.jprot.2019.103594. Epub 2019 Nov 20.
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.
糖基化是一种常见的蛋白质 PTM，其异常调节与各种病理状况（包括癌症）广泛相关。我们最近开发的完整 N-糖肽搜索引擎 GPSeeker 使我们能够使用肽骨干的同位素标记和 N-糖链部分的结构特异性片段离子，对差异表达的完整 N-糖肽（DEGPs）进行相对定量的结构特异性表征。在这里，我们报告了我们使用 RPLC-nanoESI-MS/MS 和 GPSeeker 对 MCF-7 乳腺癌细胞（相对于上皮 MCF-10A 细胞）进行的基于位置和结构的相对定量 N-糖蛋白质组学研究。在谱级 FDR≤1%的情况下，从五个技术重复（TR1、TR2、TR3、TR4 和 TR5）中鉴定出 581 个具有肽骨干（氨基酸序列、N-糖基位点）和 N-糖链部分（单糖组成、序列和连接）综合结构信息的完整 N-糖肽。使用定量至少三次的标准，其中 5 个技术重复的变化小于 1.5 倍，p≤0.05 和 RSD≤20%，从 19 个完整 N-糖蛋白的 23 个 N-糖基位点定量了 56 个 DEGPs。对于观察到差异 N-糖基化表达的 19 个完整 N-糖蛋白，14 个（每个都有一个或多个 DEGPs）观察到均匀下调；6 个（每个都有一个或多个 DEGPs）观察到均匀上调；而一个同时观察到上调和下调。意义：使用 RPLC-nanoESI-MS/MS（带有分步 NCE 的 HCD）和完整 N-糖肽搜索引擎 GPSeeker，通过基于位置和结构的 N-糖蛋白质组学，对 MCF-7 乳腺癌细胞（相对于 MCF-10A 上皮细胞）的差异 N-糖基化进行了定性和定量表征。在谱级 FDR≤1%的情况下，鉴定出 581 个具有肽骨干和 N-糖链部分综合结构信息的完整 N-糖肽；对于 248 个假定的 N-糖基位点，248 个得到了确认，其中 125 个截至 2019 年 7 月 25 日尚未在 UniProt 中注释。对于 114 个 N-糖假定连接结构，有 44 个得到了确认，至少有一个结构诊断性片段离子。使用定量至少三次的标准，其中 5 个技术重复的变化小于 1.5 倍，p≤0.05，从 21 个完整 N-糖蛋白中定量了 56 个 DEGPs；13 个和 5 个完整 N-糖蛋白（每个都有一个或多个 DEGPs）观察到均匀下调和上调；而一个同时观察到上调和下调。
