Abstract
Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity.
Highlights
Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins
We evaluated the performance of the described complex-centric analysis method, (i) by benchmarking the CCprofiler algorithm and error model against a manually curated reference dataset, (ii) by comparing its performance with the performance of a reference method consisting of multidimensional co-fractionation of native complexes and the proteomic analysis of 1,163 fractions by datadependent mass spectrometry (Havugimana et al, 2012), and (iii) by demonstrating increased sensitivity for complex detection as a result of the improved consistency of quantification of SWATH/DIA compared to data-dependent acquisition-based mass spectrometry (Fig 2)
The results show that the complex-centric analysis method, without direct use of CORUM priors, recalls 565 complexes from 81 fractions generated by single-dimensional size exclusion chromatography (SEC), compared to 561 complexes recalled from 1,163 fractions by multidimensional fractionation (Havugimana et al, 2012) and 335 complexes recalled by external SEC-dependent acquisition (DDA)-MS (Larance et al, 2016; Fig 2B)
Summary
Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, of great biological and clinical significance. We describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error-controlled, complex-centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub-complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
