一种制备组蛋白质谱分析样品的新方法

A Novel Method of Preparation Samples for Mass Spectrometry Analysis of Histones

目的:质谱技术已经广泛应用于分析组蛋白的翻译后修饰。传统的样品制备是将酸提取的组蛋白混合物在溶液中酰基化并胰酶酶解,不但操作过程复杂,而且难以解释鉴定肽段的最终归属,因此有必要对其进行改进。方法:首先对组蛋白纯化试剂盒提取的组蛋白H3/H4组分进行高效液相色谱分离,然后对纯化的的单个H3.1和H4进行胶内丙酰化衍生和胰酶消化,最后将制备的样品进行质谱分析和数据库检索。结果:鉴定到的组蛋白肽段数目至少提高60%且修饰种类和位点都有不同程度的提高。结论:建立了一种简便有效的基于高效液相色谱纯化和胶内丙酰化衍生的制备组蛋白质谱分析样品的新方法。

Objective： Mass spectrometry（MS） -based techniques have been applied as a very efficient tool for deciphering post -transla-tional modification of histones. The traditional sample preparation for MS analysis included acid - extraction of bulk histones followed by in - solution acylation and trypsinolysis. This. approach was not only very complex but also hard to interpret the final attribution of identified peptides. It was necessary to improve sample preparation for MS analysis of histones. Method ： H3/H4 fraction extracted by histone purifi-cation kit was further separated by high performance liquid chromatography（HPLC）. Purified single H3.1 and H4 were in-gel propiony-lated,digested by trypsin and finally subject to MS analysis and database search. Result： The numbers of identified peptides were in-creased at least by 60% and the modification types and sites were also increased at different degrees. Conclusion： A simple and efficient method was developed for preparation histone samples for MS analysis based on HPLC purification and in - gel propionylation.