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Developing Workflow for Simultaneous Analyses of Phosphopeptides and Glycopeptides.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-01-02 , DOI: 10.1021/acschembio.8b00902
Kyung-Cho Cho 1 , Lijun Chen 1 , Yingwei Hu 1 , Michael Schnaubelt 1 , Hui Zhang 1
Affiliation  

Enrichment of modified peptides from global peptides is inevitable in mass spectrometric analysis protein modifications because of their importance in the study of cellular functions and low abundance in the global proteomic analysis. Recent advances in enrichment methods for modified peptides such as phosphopeptides and intact glycopeptides (IGPs) show that the methods for proteomic analyses of both protein modifications are robust. We have recently observed and reported a large number of IGPs from phosphoproteomic analysis using IMAC-based phosphopeptides enrichment procedure. To determine whether phosphorylated peptides could be specifically isolated from coenriched IGPs in IMAC experiments with different pH, IMAC procedures were performed at different pH conditions, and we found that the enrichment of phosphopeptides at pH 2.0 was the optimal condition for having the highest number of phosphopeptide identifications; however, coenrichment of phosphopeptides and glycopeptides was inevitable in the entire pH range. The hydrophilic enrichments of IGPs performed before or after IMAC enrichment were evaluated subsequently to determine the optimal workflow for simultaneous analyses of phosphopeptides and glycopeptides, and IMAC enrichment followed by hydrophilic enrichment was chosen as the optimized workflow. Applying the workflow to the TMT-labeled peptides from luminal and basal-like type of breast cancer patient-derived xenograft (PDX) models allowed quantitative analyses of phospho- and glycoproteomics with 17582 phosphopeptides and 3468 glycopeptides identified, and 1237 phosphopeptides and 236 glycopeptides showed significant expression differences between luminal and basal-like, respectively. This method allows simultaneous analyses of phosphoprotein and glycoprotein modifications, extending our understanding of roles of glycosylation and phosphorylation in biology and diseases.

中文翻译:

开发同时进行磷酸肽和糖肽分析的工作流程。

在质谱分析蛋白质修饰中不可避免地要从整体肽中富集修饰的肽,因为它们在细胞功能研究中的重要性和在整体蛋白质组学分析中的低丰度是很重要的。修饰肽(例如磷酸肽和完整糖肽(IGP))的富集方法的最新进展表明,两种蛋白质修饰的蛋白质组学分析方法都非常可靠。我们最近已经观察到并报道了使用基于IMAC的磷酸肽富集程序进行的磷酸化蛋白质组学分析所产生的大量IGP。为了确定是否可以在具有不同pH的IMAC实验中从共富集的IGP中特异性分离磷酸化的肽,在不同的pH条件下进行了IMAC程序,我们发现在pH 2下可以富集磷酸肽。0是具有最高数量的磷酸肽鉴定的最佳条件;然而,在整个pH范围内,磷酸肽和糖肽的共富集是不可避免的。随后评估在IMAC富集之前或之后进行的IGP的亲水富集,以确定同时分析磷酸肽和糖肽的最佳工作流程,然后选择IMAC富集随后进行亲水富集作为优化工作流程。将工作流程应用于来自患者的腔样和基底样类型的异种移植(PDX)模型的TMT标记的肽,可以对磷酸化和糖蛋白组学进行定量分析,鉴定出17582个磷酸肽和3468个糖肽,1237个磷酸肽和236个糖肽分别在腔和基底样蛋白之间表现出显着的表达差异。这种方法可以同时分析磷蛋白和糖蛋白的修饰,扩展了我们对糖基化和磷酸化在生物学和疾病中的作用的理解。
更新日期:2018-12-10
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