Jiang Xinning, Han Guanghui, Feng Shun, Jiang Xiaogang, Ye Mingliang, Yao Xuebiao, Zou Hanfa
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, China.
J Proteome Res. 2008 Apr;7(4):1640-9. doi: 10.1021/pr700675j. Epub 2008 Mar 4.
Manual checking is commonly employed to validate the phosphopeptide identifications from database searching of tandem mass spectra. It is very time-consuming and labor intensive as the number of phosphopeptide identifications increases greatly. In this study, a simple automatic validation approach was developed for phosphopeptide identification by combining consecutive stage mass spectrometry data and the target-decoy database searching strategy. Only phosphopeptides identified from both MS2 and its corresponding MS3 were accepted for further filtering, which greatly improved the reliability in phosphopeptide identification. Before database searching, the spectra were validated for charge state and neutral loss peak intensity, and then the invalid MS2/MS3 spectra were removed, which greatly reduced the database searching time. It was found that the sensitivity was significantly improved in MS2/MS3 strategy as the number of identified phosphopeptides was 2.5 times that obtained by the conventional filter-based MS2 approach. Because of the use of the target-decoy database, the false-discovery rate (FDR) of the identified phosphopeptides could be easily determined, and it was demonstrated that the determined FDR can precisely reflect the actual FDR without any manual validation stage.
手动检查通常用于验证串联质谱数据库搜索得到的磷酸化肽段鉴定结果。随着磷酸化肽段鉴定数量的大幅增加，这一过程非常耗时且 labor intensive。在本研究中，通过结合连续阶段质谱数据和目标-诱饵数据库搜索策略，开发了一种简单的自动验证方法用于磷酸化肽段鉴定。仅接受从MS2及其相应MS3中均鉴定出的磷酸化肽段进行进一步筛选，这大大提高了磷酸化肽段鉴定的可靠性。在数据库搜索之前，对谱图进行电荷状态和中性丢失峰强度验证，然后去除无效的MS2/MS3谱图，这大大减少了数据库搜索时间。结果发现，在MS2/MS3策略中，灵敏度显著提高，因为鉴定出的磷酸化肽段数量是传统基于过滤器的MS2方法的2.5倍。由于使用了目标-诱饵数据库，可以轻松确定鉴定出的磷酸化肽段的错误发现率（FDR），并且证明所确定的FDR无需任何手动验证阶段就能精确反映实际FDR。
