Ryzhaya Palina, Pírek Pavlína, Zdráhal Zbyněk, Lochmanová Gabriela
Mendel Center for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic.
Anal Chem. 2025 Jun 24;97(24):12486-12492. doi: 10.1021/acs.analchem.5c02238. Epub 2025 Jun 12.
Arginine-specific cleavage is the primary method used to prepare lysine-rich histone proteins in bottom-up proteomics. As the Arg-C enzyme has demonstrated suboptimal specificity, cleavage at the carboxyl side of arginine residues is typically achieved through the chemical derivatization of lysines followed by trypsin digestion. Recent improvements in proteolytic enzymes are reflected in the introduction of Arg-C Ultra, a recombinant proteinase with a substantially improved digestion specificity. Here, using mammalian histone extract, we demonstrate that Arg-C Ultra facilitates histone preparation for LC-MS/MS. We show the performance of Arg-C Ultra in terms of digestion specificity, number of modified forms identified, and yield of quantitative information compared with Arg-C and trypsin digestion combined with chemical derivatization with trimethylacetic anhydride. Importantly, we show that chemical derivatization at the peptide level, i.e., after Arg-C Ultra digestion, is still necessary to improve the quantification of short histone peptidoforms as well as positional isomers.
在自下而上的蛋白质组学中，精氨酸特异性切割是制备富含赖氨酸组蛋白的主要方法。由于Arg-C酶的特异性欠佳，通常通过赖氨酸的化学衍生化，然后进行胰蛋白酶消化，来实现精氨酸残基羧基侧的切割。蛋白水解酶的最新改进体现在Arg-C Ultra的引入上，它是一种重组蛋白酶，消化特异性有了显著提高。在这里，我们使用哺乳动物组蛋白提取物，证明了Arg-C Ultra有助于用于液相色谱-串联质谱（LC-MS/MS）的组蛋白制备。与Arg-C以及胰蛋白酶消化结合三甲基乙酸酐化学衍生化相比，我们展示了Arg-C Ultra在消化特异性、鉴定的修饰形式数量以及定量信息产量方面的性能。重要的是，我们表明，在肽水平上进行化学衍生化，即在Arg-C Ultra消化后，对于改善短组蛋白肽形式以及位置异构体的定量仍然是必要的。
