Chemical derivatization of histones for facilitated analysis by mass spectrometry
Chemical derivatization of histones for facilitated analysis by mass spectrometry
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DOI：
10.1038/nprot.2007.106
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Hunt, Donald F.
中科院分区：
文献类型：
--
作者：
Garcia, Benjamin A.;Mollah, Sahana;Hunt, Donald F.
组蛋白翻译后修饰由于其在调节基因表达中的作用，近年来得到了广泛的研究。在这里，我们描述了使用化学衍生化和串联质谱法定性和半定量方式表征组蛋白修饰的方案。在这些程序中，提取的组蛋白首先用丙酸酐衍生化，以中和电荷和阻断赖氨酸残基，然后用胰蛋白酶消化，在这些条件下，胰蛋白酶只切割精氨酸残基。生成的肽可以很容易地使用在线lc -电喷雾电离-串联质谱分析来确定修饰位点。此外，一个稳定的同位素标记步骤可以包括修改羧酸基团允许组蛋白修饰的相对量化。这种方法的优点是从高度修饰的蛋白质中产生少量预测肽。该方案约需15-19小时完成，包括所有化学反应、酶消化和质谱实验。
Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments.