Pesavento James J, Garcia Benjamin A, Streeky James A, Kelleher Neil L, Mizzen Craig A
Center for Biophysics and Computational Biology, Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Mol Cell Proteomics. 2007 Sep;6(9):1510-26. doi: 10.1074/mcp.M600404-MCP200. Epub 2007 Jun 14.
Recent developments in top down mass spectrometry have enabled closely related histone variants and their modified forms to be identified and quantitated with unprecedented precision, facilitating efforts to better understand how histones contribute to the epigenetic regulation of gene transcription and other nuclear processes. It is therefore crucial that intact MS profiles accurately reflect the levels of variants and modified forms present in a given cell type or cell state for the full benefit of such efforts to be realized. Here we show that partial oxidation of Met and Cys residues in histone samples prepared by conventional methods, together with oxidation that can accrue during storage or during chip-based automated nanoflow electrospray ionization, confounds MS analysis by altering the intact MS profile as well as hindering posttranslational modification localization after MS/MS. We also describe an optimized performic acid oxidation procedure that circumvents these problems without catalyzing additional oxidations or altering the levels of posttranslational modifications common in histones. MS and MS/MS of HeLa cell core histones confirmed that Met and Cys were the only residues oxidized and that complete oxidation restored true intact abundance ratios and significantly enhanced MS/MS data quality. This allowed for the unequivocal detection, at the intact molecule level, of novel combinatorially modified forms of H4 that would have been missed otherwise. Oxidation also enhanced the separation of human core histones by reverse phase chromatography and decreased the levels of salt-adducted forms observed in ESI-FTMS. This method represents a simple and easily automated means for enhancing the accuracy and sensitivity of top down analyses of combinatorially modified forms of histones that may also be of benefit for top down or bottom up analyses of other proteins.
自上而下质谱分析技术的最新进展，使得人们能够以前所未有的精度识别和定量密切相关的组蛋白变体及其修饰形式，有助于更好地理解组蛋白如何促进基因转录及其他核过程的表观遗传调控。因此，为充分实现这些研究的价值，完整的质谱图谱准确反映给定细胞类型或细胞状态中存在的变体和修饰形式的水平至关重要。在此，我们表明，传统方法制备的组蛋白样品中蛋氨酸（Met）和半胱氨酸（Cys）残基的部分氧化，以及储存过程中或基于芯片的自动纳升电喷雾电离过程中可能发生的氧化，会改变完整质谱图谱，并阻碍串联质谱（MS/MS）后翻译修饰定位，从而混淆质谱分析。我们还描述了一种优化的过甲酸氧化程序，该程序可避免这些问题，且不会催化额外的氧化反应或改变组蛋白中常见的翻译后修饰水平。对HeLa细胞核组蛋白进行的质谱和串联质谱分析证实，Met和Cys是仅被氧化的残基，完全氧化可恢复真正的完整丰度比，并显著提高串联质谱数据质量。这使得在完整分子水平上能够明确检测到新型的H4组合修饰形式，否则这些形式会被遗漏。氧化还增强了人核心组蛋白在反相色谱中的分离效果，并降低了电喷雾傅里叶变换质谱（ESI-FTMS）中观察到的盐加合物形式的水平。该方法代表了一种简单且易于自动化的手段，可提高对组蛋白组合修饰形式进行自上而下分析的准确性和灵敏度，这对于其他蛋白质的自上而下或自下而上分析也可能有益。
