蛋白质甲基化动态修饰的深度覆盖定量分析新方法及在上皮细胞间质转化过程中的调控机制研究

Protein methylation is closely related to cancer, AIDS and cardiovascular disease. Because of its low abundance and little change in the physical and chemical properties of amino acids, the enrichment efficiency and the quantitative accuracy of current method is poor. According to the above problems, this project is intended to use the specific enzyme Lysarginase to cleave the N-terminus of protein lysine and arginine with methylation or without methylation, whereas trypsin can not be used for methylated Lysine and arginine digestion. Combined with biotin labeling the N-terminal of the non-methylated polypeptide and removing by using affinity column, a new method for high efficiency enrichment of methylation was established to improve enrichment selectivity of protein methylation modification as well as to enrich multiple type’s methylation at the same time. DDA scanning mode of mass spectrometry and SWATH acquisition mode were combined to establish a new method for quantitative analysis of methylation to improve the reproducibility and accuracy of quantitative analysis. The established method is used for dynamic protein methylation analysis of TGF-β-induced lung cancer A549 cells epithelia-mesenchymal transition and find that EMT-related protein methylation sites to reveal its regulation mechanism in the EMT process initially.

蛋白质甲基化动态修饰与癌症、艾滋病以及心血管疾病等密切相关，其丰度低，对氨基酸理化性质改变小，并且同一位点有多种类型甲基化修饰，现有分析方法富集选择性差和定量准确性低。针对上述问题本项目拟开展：利用特异性酶Lysarginase可以对蛋白质甲基化和无甲基化修饰的赖氨酸和精氨酸的N端进行酶切，而胰蛋白酶无法对甲基化修饰的赖氨酸和精氨酸酶切的特性，结合生物素标记对无甲基化多肽N端进行标记，利用亲和作用的原理将其去除，建立甲基化修饰的高选择性富集新方法，提高甲基化修饰的富集选择性，并且实现多种甲基化修饰的同时富集；将DDA质谱扫描模式及SWATH采集模式相结合，建立甲基化动态修饰分析的无标定量新方法，提高定量分析的重现性和准确度；将建立的方法用于TGF-β诱导肺癌A549细胞发生EMT过程中蛋白质甲基化动态修饰分析，发现与EMT发生相关的蛋白质甲基化修饰位点，初步揭示其在EMT过程的调控机制。

针对目前蛋白质甲基化修饰分析过程中富集选择性差和定量准确性低等问题，发展了多类型蛋白质甲基化修饰反向富集分析新方法，大大提高了蛋白质甲基化修饰的富集效率，利用该策略，可从Hela细胞中鉴定到1148个甲基化修饰类型；为了进一步提高富集效率和简化方法流程，发展了LysargNase定点释放多种类型蛋白质甲基化修饰肽段的分析新方法，可从Hela细胞中在1012蛋白质上，鉴定出1585个甲基化修饰位点信息，其中包括212个赖氨酸单甲基化修饰位点、260个赖氨酸二甲基化修饰位点、295个赖氨酸三甲基化修饰位点、414个精氨酸单甲基化修饰位点和404个精氨酸二甲基化修饰位点；建立了一种基于碎片离子及放大窗口的蛋白质组多重定量方法，实现了六个样品的同时分析，并且在20倍动态范围内，蛋白质定量结果的平均相对误差为10%。将建立的新方法用于TGF-β诱导的肺癌A549细胞EMT过程中蛋白质甲基化修饰的动态变化分析，发现了17个甲基化修饰位点发生了变化。揭示了TGFβ1作用于细胞后与其受体结合，通过使RNA结合蛋白发生甲基化修饰，改变其与RNA的结合能力，从而调控了mRNA加工，剪切，代谢等转录后调控过程，从而影响上皮细胞的关键蛋白如E-cadherin表达下调，间质细胞关键蛋白如Vimentin等表达上调，从而最终导致EMT发生。相关研究成果发表论文6篇，其中SCI论文4篇，申请发明专利4项，培养博士研究生1名，博士后1名。