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.
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.
Banting and Best Department of Medical Research, University of Toronto, 160 College St., Toronto, Ontario, Canada M5S 3E1.
Cuihong
C
Wan
Jian
J
Liu
Vincent
V
Fong
Andrew
A
Lugowski
Snejana
S
Stoilova
Dylan
D
Bethune-Waddell
Blake
B
Borgeson
Pierre C
PC
Havugimana
Edward M
EM
Marcotte
Andrew
A
Emili
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
D011506:Proteins