期刊
JOURNAL OF PROTEOME RESEARCH
卷 2, 期 1, 页码 43-50出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr025556v
关键词
proteome; tandem mass spectrometry; LC-MS/MS; vented column; Sequest criteria
资金
- NHGRI NIH HHS [HG00041] Funding Source: Medline
Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 mum i.d.) approach for RP-LC-MS/MS analysis. More than 162 000 MS/MS spectra were collected with 26 815 matched to yeast peptides (7537 unique pepticles). A total of 1504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J.R., III. Nat. Biotechnol. 2001, 19,242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.
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