Analysis of the Proteome of Saccharomyces cerevisiae for Methylarginine

Arginine methylation is a post-translational modification that has been implicated in a plethora of cellular processes. In the present manuscript, using two antimethylarginine antibodies and combinatorial deletion mutants of arginine methyltransferases, we found evidence of widespread arginine methylation in the Saccharomyces cerevisiae proteome. Immunoprecipitation was used for enrichment of methylarginine-containing proteins, which were identified via tandem mass spectrometry. From this, we identified a total of 90 proteins, of which 5 were previously known to be methylated. The proteins identified were involved in known methylarginine-associated biological functions such as RNA processing, nuclear transport, carbohydrate metabolic process, GMP biosynthetic process and protein folding. Through in vivo methylation by the incorporation of [3H]-methyl groups, we validated the methylation of 7 proteins (Ded1, Imd4, Lhp1, Nop1, Cdc11, Gus1, Pob3). By LC–MS/MS, we then confirmed a total of 15 novel methylarginine sites on 5 proteins (Ded1, Lhp1, Nop1, Pab1, and Ugp1). By examination of methylation on proteins from the triple knockout of methyltransferases Hmt1, Hsl7, Rmt2, we present evidence for the existence of additional unidentified arginine methyltransferases in the Saccharomyces cerevisiae proteome.精氨酸甲基化（Arginine methylation）是一种翻译后修饰（post-translational modification），已被证实参与大量细胞过程。本研究采用两种抗甲基精氨酸抗体（antimethylarginine antibodies），结合精氨酸甲基转移酶（arginine methyltransferases）组合缺失突变体，在酿酒酵母（Saccharomyces cerevisiae）蛋白质组（proteome）中发现了广泛存在的精氨酸甲基化现象。我们通过免疫沉淀（Immunoprecipitation）法富集含甲基精氨酸的蛋白质，并借助串联质谱（tandem mass spectrometry）对其进行鉴定。本次鉴定共得到90种蛋白质，其中5种此前已被报道存在甲基化修饰。所鉴定的蛋白质参与了已知的与精氨酸甲基化相关的生物学过程，包括RNA加工、核运输、碳水化合物代谢、GMP生物合成以及蛋白质折叠等。通过掺入[3H]-甲基基团进行体内甲基化实验，我们验证了7种蛋白质的甲基化修饰，分别为Ded1、Imd4、Lhp1、Nop1、Cdc11、Gus1与Pob3。随后通过液相色谱-串联质谱（LC–MS/MS），我们在5种蛋白质（Ded1、Lhp1、Nop1、Pab1及Ugp1）上确认了共计15个全新的精氨酸甲基化位点。通过对精氨酸甲基转移酶Hmt1、Hsl7和Rmt2三基因敲除菌株的蛋白质甲基化水平进行检测，我们发现酿酒酵母蛋白质组中还存在其他未被鉴定的精氨酸甲基转移酶。