一种适用于质谱分析的简化胶内酶解方法

A Simple Protein In-Gel Digest Method Compatible with Mass Spectrometry Analysis

在常规方法的基础上,设计了一种适于质谱分析的简化胶内酶解方法。改进的步骤包括:(1)通过加大洗涤所用超纯水量、延长涡混时间来强化凝胶洗涤的环节;(2)加入酸化处理来提高胰蛋白酶的活性;(3)在预酶解时不加CaCl_2,减少了酶的自切作用;(4)省略了蛋白质样品脱盐、脱SDS的步骤;(5)直接吸取酶解液进行质谱分析。系统比较该简化酶解法和最新报道的一种酶解方法的质谱鉴定效果,简化法能有效减少酶解后肽段的损失,增加质谱数据库搜索的信息量,得到更可靠的蛋白质鉴定结果。

A simple protein in-gel digest method compatible with MALDI-TOF MS analysis was developed from the previously reported protocols with the following modifications. （1） The washing step was intensified by increasing the volume of distilled water and prolonging the vortex time. （2） Trypsin was acidified to improve the enzyme activity. （3） A Ca^2＋ free pre-digest solution was used to reduce the trypsin auto cleavage. （4） The procedure of removal of salts and SDS was cancelled. （5） The digest was directly used to per- form MALDI-TOF MS analysis. The compared results of this method with a latest reported protocol demonstrated that the method could efficientlyreduce the peptide loss, provide more information for MS analysis, and consequently make the protein identification more reliable.