超声波辅助Urea-Free试剂结合热变性快速酶解蛋白质  

Ultrasound-assisted Urea-Free Chemical Denaturants Combined with Thermal Denaturation to Accelerate Enzymatic Proteolysis

作　　者：李雅萍[1] 李友元[1] 周新文[2] 陈长华[1] 张嗣良[1] 杨芃原[2] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]复旦大学生物医学研究院,上海200032

出　　处：《分析化学》2010年第5期663-667,共5页Chinese Journal of Analytical Chemistry

基　　金：国家科技支撑计划项目(No.2008BAI63B01);生物反应器工程国家重点实验室专项经费(No.2060204)资助

摘　　要：建立了一个超声功率-超声时间的安全区域,在此范围内进行超声波辅助酶解时,能避免超声波随机断裂蛋白质;在此安全区域内采用响应面法获得最佳超声功率和超声时间。建立了超声波辅助Urea-Free试剂结合热变性快速酶解蛋白质的方法,将蛋白质依次进行10min超声波浴辅助Urea-Free还原烷基化反应、90℃热变性15min,超声波辅助胰蛋白酶酶解15s。应用此快速酶解方法鉴定3种典型标准蛋白质(牛血清白蛋白、细胞色素C和肌红蛋白),在25min内达到与传统整夜酶解方法相同的鉴定结果,并获得更高的序列覆盖率。A safe range of ultrasonication power-time in which ultrasound would not break protein at random was established. The optimum ultrasonication parameters were obtained by response surface design method. Sequentially, an ultrasound bath-assisted method of urea-free chemical denaturants combined with thermal denaturation to accelerate enzymatic proteolysis was set up. First, protein reduction and alkylation were done in 10 min without urea with the aid of ultrasound bath, then following with thermal denaturation at 90 ℃ for 15 min and successful in-solution ultrasound-assisted tryptic proteolysis in 15 s. Compared with conventional overnight incubation method, three typical proteins-bovine serum albumin, cytochrome C and myoglobin were successfully identified in 25 min by the ultrasonic-assisted protein digestion procedure with equal digestion efficiency and higher sequence coverage.

关 键 词：超声波辅助 热变性 蛋白质 酶解 质谱 

分 类 号：Q51[生物学—生物化学]