Baumgartner Christian, Rejtar Tomas, Kullolli Majlinda, Akella Lakshmi Manohar, Karger Barry L
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
J Proteome Res. 2008 Sep;7(9):4199-208. doi: 10.1021/pr800277y. Epub 2008 Aug 8.
A novel computational approach, termed Search for Modified Peptides (SeMoP), for the unrestricted discovery and verification of peptide modifications in shotgun proteomic experiments using low resolution ion trap MS/MS spectra is presented. Various peptide modifications, including post-translational modifications, sequence polymorphisms, as well as sample handling-induced changes, can be identified using this approach. SeMoP utilizes a three-step strategy: (1) a standard database search to identify proteins in a sample; (2) an unrestricted search for modifications using a newly developed algorithm; and (3) a second standard database search targeted to specific modifications found using the unrestricted search. This targeted approach provides verification of discovered modifications and, due to increased sensitivity, a general increase in the number of peptides with the specific modification. The feasibility of the overall strategy has been first demonstrated in the analysis of 65 plasma proteins. Various sample handling induced modifications, such as beta-elimination of disulfide bridges and pyrocarbamidomethylation, as well as biologically induced modifications, such as phosphorylation and methylation, have been detected. A subsequent targeted Sequest search has been used to verify selected modifications, and a 4-fold increase in the number of modified peptides was obtained. In a second application, 1367 proteins of a cervical cancer cell line were processed, leading to detection of several novel amino acid substitutions. By conducting the search against a database of peptides derived from proteins with decoy sequences, a false discovery rate of less than 5% for the unrestricted search resulted. SeMoP is shown to be an effective and easily implemented approach for the discovery and verification of peptide modifications.
本文提出了一种名为“修饰肽搜索（SeMoP）”的新型计算方法，用于在使用低分辨率离子阱MS/MS谱的鸟枪法蛋白质组学实验中无限制地发现和验证肽修饰。使用这种方法可以识别各种肽修饰，包括翻译后修饰、序列多态性以及样品处理引起的变化。SeMoP采用三步策略：（1）通过标准数据库搜索识别样品中的蛋白质；（2）使用新开发的算法无限制地搜索修饰；（3）针对无限制搜索中发现的特定修饰进行第二次标准数据库搜索。这种靶向方法可验证发现的修饰，并且由于灵敏度提高，具有特定修饰的肽数量总体增加。整体策略的可行性首先在对65种血浆蛋白的分析中得到证明。已检测到各种样品处理引起的修饰，如二硫键的β-消除和焦碳酰胺甲基化，以及生物诱导的修饰，如磷酸化和甲基化。随后使用靶向的Sequest搜索来验证选定的修饰，修饰肽的数量增加了4倍。在第二个应用中，对一种宫颈癌细胞系的1367种蛋白质进行了处理，从而检测到了几种新的氨基酸取代。通过对来自具有诱饵序列的蛋白质的肽数据库进行搜索，无限制搜索的错误发现率低于5%。结果表明，SeMoP是一种用于发现和验证肽修饰的有效且易于实施的方法。
