ComplexQuant: high-throughput computational pipeline for the global quantitative analysis of endogenous soluble protein complexes using high resolution protein HPLC and precision label-free LC/MS/MS

Autor: Vincent Fong, Jian Liu, Pierre C. Havugimana, Snejana Stoilova, Dylan Bethune-Waddell, Andrew Lugowski, Cuihong Wan, Blake Borgeson, Andrew Emili, Edward M. Marcotte
Rok vydání: 2012
Předmět:
Zdroj: Journal of proteomics. 81
ISSN: 1876-7737
Popis: 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: From protein structures to clinical applications.
Databáze: OpenAIRE
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