Dormeyer Wilma, Ott Melanie, Schnölzer Martina
Protein Analysis Facility, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany.
Mol Cell Proteomics. 2005 Sep;4(9):1226-39. doi: 10.1074/mcp.M500047-MCP200. Epub 2005 Jun 2.
The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-enzymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/CBP-associated factor (PCAF).
乙酰转移酶对特定赖氨酸残基进行的蛋白质乙酰化已成为一种具有高度生物学影响的翻译后修饰。尽管组蛋白中的赖氨酸乙酰化是组蛋白密码的一个组成部分，但最近人们认识到许多非组蛋白的乙酰化在许多细胞过程中是一种调节信号。乙酰转移酶的新底物不断被鉴定出来，蛋白质中乙酰化位点的分析越来越多地通过质谱法进行。然而，使用质谱技术对蛋白质中的赖氨酸乙酰化进行表征存在一些局限性和陷阱。特别是非酶促的半胱氨酸乙酰化可能导致乙酰化蛋白质的假阳性鉴定。在这里，我们展示了各种质谱技术，如基质辅助激光解吸/电离和电喷雾电离质谱在蛋白质乙酰化分析中的应用。我们描述了用于绘制蛋白质中乙酰化位点的多种生化方法组合，并讨论了它们的优缺点。作为一个例子，我们对HIV-1转录反式激活因子（Tat）蛋白的乙酰化进行了详细分析，已知该蛋白在体内被乙酰转移酶p300和p300/CBP相关因子（PCAF）乙酰化。
