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Complex-centric proteome profiling by SEC-SWATH-MS.

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.

SUBMITTER: Heusel M

PROVIDER: S-EPMC6346213 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Complex-centric proteome profiling by SEC-SWATH-MS.

Heusel Moritz M Bludau Isabell I Rosenberger George G Hafen Robin R Frank Max M Banaei-Esfahani Amir A van Drogen Audrey A Collins Ben C BC Gstaiger Matthias M Aebersold Ruedi R

Molecular systems biology 20190114 1

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 consist ...[more]

PMID: 30642884

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Project description:Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract.

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Complex-centric proteome profiling by SEC-SWATH-MS.

Publications

Complex-centric proteome profiling by SEC-SWATH-MS.

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