Proteomic Analysis of Protein Methylation in the Yeast Saccharomyces cerevisiae

Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the SAM-auxotrophic Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 117 methylated proteins, containing 182 methylation events associated with 174 methylation sites. About 90% of these methylation events were previously unknown. Our results indicated, 1) over 6% of the yeast proteome are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys >> Arg > Asp > Glu ≈ Gln ≈ Asp > His > Cys, 3) the methylation state on nitrogen center is largely exclusive, and 4) the methylated proteins are located mainly in nucleus/ribosome associated with translation/transcription and DNA/RNA processing. Our dataset is the most comprehensive methylproteome known-to-date of all living organisms, and should significantly contribute to the field of protein methylation and related research.

由依赖S-腺苷甲硫氨酸（S-adenosylmethionine, SAM）的甲基转移酶催化的蛋白质甲基化，是一类参与诸多重要生物学过程的关键翻译后修饰（Post-translational modification, PTM）。由于甲基化可发生于氮、氧、硫原子位点，且氮原子位点存在多种甲基化状态，因此甲基化蛋白质组（methylproteome）的相关研究仍较为匮乏。本研究基于在线多维μHPLC/MS/MS技术，建立了同位素标记SAM法（Methylation by Isotope Labeled SAM, MILS），用于对SAM营养缺陷型酿酒酵母（SAM-auxotrophic Saccharomyces cerevisiae）的甲基化蛋白质组进行高可信度分析。本研究共鉴定得到117个甲基化蛋白质，涵盖182个甲基化事件，对应174个甲基化位点。其中约90%的甲基化事件为此前未被报道的新发现。我们的研究结果表明：1）酿酒酵母蛋白质组中超过6%的蛋白发生甲基化修饰；2）蛋白质甲基化的氨基酸残基偏好性遵循如下顺序：赖氨酸（Lys）>> 精氨酸（Arg）> 天冬氨酸（Asp）> 谷氨酸（Glu）≈ 谷氨酰胺（Gln）≈ 天冬氨酸 > 组氨酸（His）> 半胱氨酸（Cys）；3）氮原子位点的甲基化状态基本呈排他性；4）甲基化蛋白质主要定位于细胞核/核糖体，参与翻译、转录及DNA/RNA加工过程。本数据集是目前已知所有生命体中最为全面的甲基化蛋白质组数据集，将为蛋白质甲基化及其相关研究领域提供重要支撑。