蛋白质的肽质谱指纹图分析方法的优化.pdfVIP

ISSN　1007-7626 2005 12 CN　11-3870 Q Chinese Journal of Biochemistry and Molecular Biolo y 21(6):831 ～ 839 ·技术与方法· ＊ 周新文, 　张　玲, 　谢锦云, 　陈　平, 　梁宋平 (湖南师范大学生命科学学院, 蛋白质化学研究室, 长沙　410081) 　肽质谱指纹图分析是一种常用 蛋白质 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 酶解肽段 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 浓缩体积控制在5 μl 以下和采用 10 mmol L 柠檬酸铵 缓冲液板上脱盐能提高蛋白质鉴定 准确度;在点样 时候, 采用先点样品再点基质 方法能明显 提高匹配肽段 个数和信噪比.这些优化 样品制备方法明显地提高了MALDI-TOF 质谱肽质谱指 纹图分析方法鉴定蛋白质 可靠性. 　MALDI-TOF-MS, 肽质谱指纹图分析, 样品制备 　Q51 Optimization of the Method for PeptideMass Fingerpriting Analysis of Proteins ＊ ZHOU Xin-Wen , ZHANG Lin , XIE Jin-Yun, CHEN Pin , LIANG Son -Pin (College of Lif e Sciences, Hunan Normal University , Laboratory of Protein Chemistry, Changsha 410081, Hunan, China) Abstract　Peptide mass fin erprintin analysis of proteins is the most widely used method for protein identification.To maximize the sequence covera e percenta e and improve the accuracy of protein MALDI- TOF-PMF identification, different procedures of tryptic peptide lyophilization, desaltin and spottin were tested and optimizedwith six standard proteins.It was found that remainnin of about 5 μl lyophilized tryptic di est samples, usin 10 mmol L diammonium citrate for washin matrix sample spots could si nificantly improve the accuracy of protein identification.By applyin 1 μl tryptic di est on steel tar et followed 1 μl HCCA could also increase the si nal noise ratio and sensitivity.These optimized protocols were proved to si nificantly improve the reliability of MALDI-TOF