非组蛋白甲基化的蛋白质组分析新方法研究

Protein methylation is an important post-translational-modification (PTM) that plays crucial roles in diverse physiological and pathological processes. The high-throughput and reliable identification of the methylation sites is crucial to the systematic investigation of methylation regulated biological processes. However, the methylated peptides are difficult to be efficiently isolated from complex biological sample. Besides, methylation can occur on as many as eight amino acid residues and covers eleven methylation forms, making it challenging to be processed by using traditional database searching approach. To solve above problems, several methods are to be developed in this project in the following aspects: 1) methylated peptides are enriched by utilizing chromatography based separation to remove the interference peptides. 2) in-silico demethylation process are conducted to deduce the fragment spectra corresponding to the unmodified peptides and enable the identification of peptides sequence without setting methylation as variable modifications and thus greatly facilitate the database searching process. The developed approach can be feasibly applied to the simultaneous analysis of multiple methylation forms. 3) based on the hM-SILAC and dimethyl labeling at protein level, the absolute stoichiometry of lysine methylation are determined on a large scale. Furthermore, the newly developed methods are utilized to the analysis of protein methylation in drug-resistance liver cancer cells, which could reveal the connections between protein methylation and drug-resistance.

蛋白质的甲基化修饰是一种在细胞中发挥重要调控作用的翻译后修饰。近年来随着质谱技术的发展，在蛋白质磷酸化、糖基化等高丰度的修饰分析方面取得了很大的进步。但是对于蛋白质甲基化等低丰度翻译后修饰的高灵敏度、高可信分析仍然面临重大挑战。本项目主要针对非组蛋白质甲基化修饰分析中存在的问题，发展相关的新技术、新方法，旨在提高甲基化蛋白质组的定性、定量分析能力。针对甲基化蛋白质丰度低、富集困难的问题，发展高特异性的甲基化富集和分析方法，实现甲基化蛋白质组的高效、高灵敏度解析。针对利用设可变修饰的传统数据库检索软件鉴定甲基化位点的方法中检索空间大，假阳性高的问题，发展新型数据处理策略，实现多种甲基化类型的同时鉴定。针对蛋白质甲基化动态研究缺乏很好的定量分析方法的问题，拟通过化学标记的方法发展甲基化绝对占有率的定量分析新方法，实现甲基化位点在占有率层次的差异分析。此外，还将开展相关新技术的应用研究。

针对目前非组蛋白甲基化定性、定量分析存在的技术瓶颈，开展了甲基化蛋白质组分离鉴定和定量分析新技术新方法的研究，有效提高了我国在蛋白质甲基化修饰谱方面的研究水平，形成了一个甲基化蛋白质组新技术新方法的研究团队。主要研究成果包括建立了蛋白质甲基化规模化鉴定分析新技术，精氨酸二甲基化富集分析新方法，以及细胞耐药性相关的甲基化蛋白质组定量研究技术等。特别是利用固定铜离子去除含组氨酸干扰肽，而发展起来的基于强阳离子交换色谱的甲基化肽段富集方法一共鉴定到了765个甲基化位点，包括860种不同的甲基化形式。其中27.21%的数据是赖氨酸甲基化，该比例远高于以往任何可以同时鉴定5种甲基化形式的基于色谱的实验方法。这些技术和方法大大提升了甲基化蛋白质组学分析的灵敏度、分析通量和定量准确性，为蛋白质甲基化相关的研究提供了新的技术体系和平台。