期刊
JOURNAL OF PROTEOME RESEARCH
卷 4, 期 1, 页码 53-62出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr0498638
关键词
proteomics; tandem mass spectrometry; the human proteome; LC-MS/MS; reversed database; SEQUEST criteria
资金
- NATIONAL CENTER FOR RESEARCH RESOURCES [P41RR018522] Funding Source: NIH RePORTER
- NCRR NIH HHS [RR18522] Funding Source: Medline
Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/ MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC-MS/MS analyses. The MS/ MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, suggesting that the false positive rates are significantly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039-1048), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242-247). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications from mammalian proteomes without greatly decreasing the number of identifications.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据