蛋白质的肽质谱指纹图分析方法的优化

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.

Optimization of the Method for Peptide Mass Fingerpriting Analysis of Proteins

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 protein 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.