Nonredundant mass spectrometry: A strategy to integrate mass spectrometry acquisition and analysis
Nonredundant mass spectrometry: A strategy to integrate mass spectrometry acquisition and analysis
复制标题
DOI：
10.1002/pmic.200300673
复制
发表时间：
2004-04-01
期刊：
影响因子：
3.4
通讯作者：
Corthals, GL
中科院分区：
文献类型：
--
作者：
Scherl, A;Francois, P;Corthals, GL
使用自动化数据依赖串联质谱（MIS/MS）进行蛋白质鉴定现在是标准程序。然而，在许多情况下，数据依赖性采集成为冗余采集，因为来自相同蛋白质的许多不同肽被片段化，而仅需要少数肽进行明确的鉴定。为了提高信息的质量，但减少信息量，非冗余MS（nrMS）的策略已经开发出来。使用nrMS，数据分析是整体MS采集和分析的组成部分，而不是通常执行的终点。在该nrMS工作流程中，使用基质辅助激光解吸/电离-飞行时间-飞行时间（MALDI-TOF/TOF）仪器。检索MS和限制性MS/MS数据，并在计算机消化后使用鉴定的蛋白质生成“排除列表”。然后将肽片段化仅限于排除列表中不存在的最强离子。重复该过程，直到所有峰都被考虑或样品被消耗。与nanoLC-MS/MS相比，nrMS对6个合并的二维电泳（2-DE）斑点的分析结果相似。与用于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶带分析的标准数据依赖性MALDI-MS/MS相比，nrMS显著增加了所鉴定蛋白质的数量。还发现，这种新的工作流程通过鉴定可能由翻译后修饰引起的意外肽，显著增加了序列覆盖率。
Protein identification using automated data-dependent tandem mass spectrometry (MIS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.