期刊
JOURNAL OF PROTEOMICS
卷 81, 期 -, 页码 102-111出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jprot.2012.10.001
关键词
Protein complex; Biochemical fractionation; HPLC co-elution; LC/MS/MS; Label-free quantification; Protein-protein interaction
资金
- Ontario Ministry of Research and Innovation
- Natural Sciences and Engineering Research Council of Canada (NSERC)
The experimental isolation and characterization of stable multi-protein complexes are essential to understanding the molecular systems biology of a cell. To this end, we have developed a high-throughput proteomic platform for the systematic identification of native protein complexes based on extensive fractionation of soluble protein extracts by multi-bed ion exchange high performance liquid chromatography (IEX-HPLC) combined with exhaustive label-free LC/MS/MS shotgun profiling. To support these studies, we have built a companion data analysis software pipeline, termed ComplexQuant Proteins present in the hundreds of fractions typically collected per experiment are first identified by exhaustively interrogating MS/MS spectra using multiple database search engines within an integrative probabilistic framework, while accounting for possible post-translation modifications. Protein abundance is then measured across the fractions based on normalized total spectral counts and precursor ion intensities using a dedicated tool, PepQuant. This analysis allows co-complex membership to be inferred based on the similarity of extracted protein co-elution profiles. Each computational step has been optimized for processing large-scale biochemical fractionation datasets, and the reliability of the integrated pipeline has been benchmarked extensively. This article is part of a Special Issue entitled: Prom protein structures to clinical applications. (C) 2012 Elsevier B.V. All rights reserved.
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