Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic Interaction Chromatography for Large-Scale Profiling of Protein Methylation
Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic Interaction Chromatography for Large-Scale Profiling of Protein Methylation
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基于去糖基化、多蛋白酶和亲水相互作用色谱的策略，用于大规模蛋白质甲基化分析
DOI：
10.1021/acs.analchem.7b03673
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发表时间：
2017
影响因子：
7.4
通讯作者：
Jia Chenxi
中科院分区：
文献类型：
--
作者：
Ma Min;Zhao Xinyuan;Chen Shuo;Zhao Yingyi;Yang Lu;Feng Yu;Qin Weijie;Li Lingjun;Jia Chenxi
蛋白质的可逆甲基化调节大多数细胞过程，包括信号转导、mRNA 剪接、转录控制、DNA 修复和蛋白质易位。在分子水平上对这些生物过程的基本了解需要对甲基化蛋白质进行全面表征。甲基化通常是亚化学计量的，迄今为止，只有非常有限数量的甲基化蛋白质和位点被确定。虽然本质上碱性/亲水性的甲基化肽可以通过亲水相互作用液相色谱 (HILIC) 进行富集，但其他亲水性肽可以在富集过程中共洗脱并抑制甲基化肽的检测。此外，修饰的精氨酸和赖氨酸残基不能被鸟枪式蛋白质组学中最常用的酶胰蛋白酶有效切割。为了克服这些问题，我们开发了一种新的去糖辅助甲基化位点识别（DOMAIN）策略，该策略能够以全蛋白质组的方式直接、快速且可重复地分析蛋白质甲基化。结合多维分级分离和多蛋白酶消化，我们的方法能够在 A549 细胞中鉴定 270 种蛋白质的 573 种甲基化形式，其中包括 311 种新的甲基化形式。将该技术与稳定同位素标记定量蛋白质组学和 RNA 干扰相结合，我们确定了与蛋白质精氨酸 N-甲基转移酶 3 (PRMT3) 相关的几个假定甲基化位点的差异调节。总的来说，我们用于全面绘制甲基化位点的集成蛋白质组学工作流程可以更好地了解蛋白质甲基化，同时为生物医学研究中的全局蛋白质甲基化分析提供快速有效的方法。
Reversible methylation of proteins regulates the majority of cellular processes, including signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. A fundamental understanding of these biological processes at the molecular level requires comprehensive characterization of the methylated proteins. Methylation is often substoichiometric, and only a very limited number of methylated proteins and sites have been confidently identified to date. Although the intrinsically basic/hydrophilic methylated peptides can be enriched by the hydrophilic interaction liquid chromatography (HILIC), other hydrophilic peptides can coelute during the enrichment process and suppress the detection of methylated peptides. In addition, the modified Arg and Lys residues cannot be efficiently cleaved by trypsin, the most commonly used enzyme in shotgun proteomics. To overcome these caveats, we develop a novel de-glyco-assisted methylation site identification (DOMAIN) strategy which enables straightforward, fast, and reproducible analysis of protein methylation in a proteome-wide manner. Combining multidimensional fractionation and multiprotease digestion, our method enabled the identification of 573 methylated forms in 270 proteins, including 311 new methylation forms, in A549 cells. Combining this technique with stable isotope labeling quantitative proteomics and RNA interference, we determined the differential regulation of several putative methylated sites that are related to the protein arginineN-methyltransferase 3 (PRMT3). Collectively, our integrated proteomics workflow for comprehensive mapping of methylation sites enables a better understanding of protein methylation, while providing a rapid and effective approach for global protein methylation analysis in biomedical research.