蛋白质的肽质谱指纹图分析方法的优化  

Optimization of the Method for Peptide Mass Fingerpriting Analysis of Proteins

作　　者：周新文[1] 张玲[1] 谢锦云[1] 陈平[1] 梁宋平[1] 

机构地区：[1]湖南师范大学生命科学学院,蛋白质化学研究室长沙410081

出　　处：《中国生物化学与分子生物学报》2005年第6期831-839,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划(973)项目(No.2001CB5120)~~

摘　　要：肽质谱指纹图分析是一种常用的蛋白质的鉴定方法.为了提高这种方法鉴定蛋白质时序列覆盖率和准确度,以6个标准蛋白质为分析样品,对几种不同的酶解肽段的浓缩、脱盐和点样方法进行了检验和优化.结果发现,将酶解肽段的浓缩体积控制在5μl以下和采用10mmolL柠檬酸铵缓冲液板上脱盐能提高蛋白质鉴定的准确度;在点样的时候,采用先点样品再点基质的方法能明显提高匹配肽段的个数和信噪比.这些优化的样品制备方法明显地提高了MALDITOF质谱肽质谱指纹图分析方法鉴定蛋白质的可靠性.Peptide mass fingerprinting analysis of proteins is the most widely used method for protein identification. To maximize the sequence coverage percentage and improve the accuracy of orotein MALDI-TOF-PMF identification, different procedures of tryptic peptide lyophilization, desalting and spotting were tested and optimized with six standard proteins, It was found that remainning of about 5 μl lyophilized tryptic digest samples, using 10 mmol/L diammonium citrate for washing matrix/sample spots could significantly improve the accuracy of protein identification. By applying 1 μl tryptic digest on steel target followed 1 μl HCCA could also increase the signal/noise ratio and sensitivity, These optimized protocols were proved to significantly, improve the reliability of MALDI-TOF mass peptide map analysis of proteins.

关 键 词：MALDI-TOF-MS 肽质谱指纹图分析 样品制备 蛋白质 

分 类 号：Q51[生物学—生物化学]