Quantifying in vivo, site-specific changes in protein methylation with SILAC.
Quantifying in vivo, site-specific changes in protein methylation with SILAC.
复制标题
使用 SILAC 定量体内蛋白质甲基化的位点特异性变化。
DOI：
10.1007/978-1-4939-1142-4_12
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Ong,Shao-En
中科院分区：
文献类型：
--
作者：
Lau,Ho-Tak;Lewis,KarenA;Ong,Shao-En
近年来，对蛋白质甲基化的兴趣迅速增长。基于质谱的蛋白质组学非常适合表征蛋白质修饰，但甲基化残基的多样性和缺乏有效的方法来富集甲基化蛋白质限制了蛋白质组学鉴定蛋白质甲基化位点。在这个协议中，我们比较了两种代谢标记方法，稳定同位素标记的氨基酸在细胞培养（SILAC）和它的变体重甲基SILAC，研究蛋白质甲基化。代替典型SILAC实验中的重赖氨酸和精氨酸，重甲基SILAC使用13 C，2 H甲硫氨酸作为标记氨基酸。当细胞将甲硫氨酸转化为S-腺苷甲硫氨酸时，重甲基SILAC编码每个甲基的4 Da质量标签，仅从质量差异就可以区分甲基化程度。我们提供了一个基于SILAC的蛋白质甲基化分析的协议，并强调了每种方法的优势和劣势，为有针对性的和蛋白质组学分析。
Interest in protein methylation has grown rapidly in recent years. Mass spectrometry-based proteomics is ideally suited to characterize protein modifications, but the multiplicity of methylated residues and the lack of efficient methods to enrich methylated proteins have limited the proteomic identification of protein methylation sites. In this protocol, we compare two metabolic labeling approaches, stable isotope labeling by amino acids in cell culture (SILAC) and its variant heavy methyl SILAC, for studying protein methylation. Instead of heavy lysine and arginine in the typical SILAC experiment, heavy methyl SILAC uses13C,2H methionine as the labeling amino acid. As cells convert methionine toS-adenosylmethionine, heavy methyl SILAC encodes a 4 Da mass tag for each methyl group, distinguishing between degrees of methylation is possible from mass difference alone. We provide a protocol for SILAC-based analyses of protein methylation and highlight the strengths and weaknesses of each method for targeted and proteomic analyses.