高浓度酶快速胶内酶解技术用于磷酸化蛋白质组学分析  

High Concentration Trypsin Assisted Fast In-Gel Digestion for Phosphoproteome Analysis

作　　者：刘芳洁[1,2] 叶明亮[1] 潘彦博 邹汉法[1] 

机构地区：[1]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,大连116023 [2]中国科学院研究生院,北京100049

出　　处：《分析化学》2015年第10期1452-1458,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金委创新研究群体项目(No.21321064);国家重点基础研究发展计划(No.2013CB911202)资助~~

摘　　要：聚丙烯酰胺凝胶电泳是一项高效的蛋白质分离技术,通过与质谱技术联用,可以鉴定成千上万的蛋白质点。但是,复杂繁琐的操作流程限制了它在蛋白质组学研究方面的广泛应用。本研究表明,高浓度的胰蛋白酶并不会影响胶内酶解后磷酸化肽段的富集,反而可以实现快速高效的蛋白酶解,据此建立了一种快捷、高效的蛋白质酶解方法。首先,通过聚丙烯酰胺凝胶电泳,对50μg复杂蛋白样品进行分离,分成5个组分;然后,选择高浓度胰蛋白酶酶解30 min;最后,运用固定钛离子亲和色谱小柱离心法富集磷酸化肽段。经液相色谱-串联质谱分析,成功鉴定到约2000个磷酸化位点,而传统方法则只鉴定到不足1500个位点。实验表明,在高浓度胰蛋白酶的促进作用下,胶内酶解过程在0.5 h内即可以完成,并且得到比对照组更高的磷酸化位点的鉴定量和更低的酶解漏切率,而传统方法需要16 h。这也证实了本方法不仅可以加速酶解反应,同时还能提高蛋白酶解效率。Polyacrylamide gel electrophoresis is a powerful protein separation technology. Combined with mass spectrometry,it could identify thousands of proteins in proteomics analysis. However,the time-consuming procedure restricts its applications in proteomics study. In this study,we found that high concentration trypsin did not compromise the subsequent phosphopeptide enrichment after in-gel digestion,but could promote the in-gel digestion. Hence,a new,fast and robust digestion method was established. Firstly,50 μg He La cell protein was separated into 5 fractions by SDS-PAGE,and then the proteins were digested by high concentration trypsin for 30 min. Finally,the phosphopeptides were enriched by Ti-IMAC Tip. After LC-MS / MS analysis,about 2000 phosphorylation sites were identified in the experimental group, while less than 1500 phosphorylaion sites were identified in control group. With the aid of high concentration of trypsin,in-gel digestion could be completed within only 30 min,and more phosphorylation site identifications and lower percentage of missed cleavages compared with control experiments were acquired,which demonstrated that high concentration of trypsin could not only accelerate the in-gel digestion, but also improve the phosphoproteome coverage.

关 键 词：高浓度酶 胶内酶解 磷酸化蛋白质组学 

分 类 号：O658.9[理学—分析化学] O629.73[理学—化学]