Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA.
Analyst. 2018 May 29;143(11):2508-2519. doi: 10.1039/c8an00216a.
The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.
系统的糖肽完整分析对于理解蛋白质糖基化的确切功能相关性至关重要。一个专用的工作流程，具有同时对糖肽进行特征分析和定量的能力，并且能够在特定和高通量的方式下进行，对于揭示复杂生物系统中特定的糖基化改变模式是必不可少的。在这项研究中，建立了一个增强的、专用的、大规模的糖肽完整分析的定量糖蛋白质组学工作流程，其中包括使用滤过辅助样品制备（FASP）方法改进对膜结合糖蛋白的特异性提取、使用顺序亲水相互作用液相色谱（HILIC）和多凝集素亲和（MLA）富集来增强糖肽的富集、通过 EThcD 实现糖肽的特异性特征分析、利用同量异位标记 N,N-二甲基亮氨酸（DiLeu）进行相对定量以及通过 Byonic 进行自动基于 FDR 的大规模数据分析。我们的研究首次表明，HILIC 极大地补充了 MLA 富集，只有 20%的完整 N-糖肽有重叠。当将开发的工作流程应用于 PANC1 细胞的特定糖蛋白质组学研究时，我们能够鉴定出 1067 个完整的 N-糖肽，代表 311 个糖基化位点和 88 种聚糖组成，来自 205 种糖蛋白。我们进一步应用这种方法研究 PKM2 敲除细胞与亲本乳腺癌细胞之间的糖基化改变，揭示了不同类型聚糖的 N-糖蛋白/糖肽模式和非常不同的糖基化微观异质性。为了获得更全面的糖蛋白改变图谱，还分析了用 PNGase F 处理后的糖肽。共定量了 484 个去糖基化肽，其中来自 70 种糖蛋白的 81 个去糖基化肽显示出显著变化。KEGG 途径分析显示，PI3K/Akt 信号通路高度富集，为 PKM2 敲低癌细胞依赖 Akt 激活来维持生存的先前发现提供了证据。由于糖基化是最重要的信号调节剂之一，我们的结果提供了额外的证据，表明信号通路受到代谢的密切调控。
