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      An informatic framework for decoding protein complexes by top-down mass spectrometry

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          Abstract

          Efforts to map the human protein interactome have resulted in information about hundreds to thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we combined the CORUM and UniProt databases to create candidates for an error-tolerant search engine designed for hierarchical top-down analyses, identification, and scoring of multi-proteoform complexes by native mass spectrometry.

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          Most cited references26

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          CORUM: the comprehensive resource of mammalian protein complexes—2009

          CORUM is a database that provides a manually curated repository of experimentally characterized protein complexes from mammalian organisms, mainly human (64%), mouse (16%) and rat (12%). Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The new CORUM 2.0 release encompasses 2837 protein complexes offering the largest and most comprehensive publicly available dataset of mammalian protein complexes. The CORUM dataset is built from 3198 different genes, representing ∼16% of the protein coding genes in humans. Each protein complex is described by a protein complex name, subunit composition, function as well as the literature reference that characterizes the respective protein complex. Recent developments include mapping of functional annotation to Gene Ontology terms as well as cross-references to Entrez Gene identifiers. In addition, a ‘Phylogenetic Conservation’ analysis tool was implemented that analyses the potential occurrence of orthologous protein complex subunits in mammals and other selected groups of organisms. This allows one to predict the occurrence of protein complexes in different phylogenetic groups. CORUM is freely accessible at (http://mips.helmholtz-muenchen.de/genre/proj/corum/index.html).
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            Proteome organization in a genome-reduced bacterium.

            The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.
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              Mapping Intact Protein Isoforms in Discovery Mode Using Top Down Proteomics

              A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large scale proteome analysis 1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry (MS) 2 . This “bottom up” process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous 2 characterization of alternative splice forms, diverse modifications (e.g., acetylation and methylation), and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species 3 . “Top down” interrogation of whole proteins can overcome these problems for individual proteins 4,5 , but has not been achieved on a proteome scale due to the lack of intact protein fractionation methods that are well integrated with tandem MS. Here we show, using a new four dimensional (4D) separation system, identification of 1,043 gene products from human cells that are dispersed into >3,000 protein species created by post-translational modification, RNA splicing, and proteolysis. The overall system produced >20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply-modified species in response to accelerated cellular aging (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database 6 , the data provide precise correlations to individual genes and proof-of-concept for large scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research 7 .
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                24 December 2015
                18 January 2016
                March 2016
                18 July 2016
                : 13
                : 3
                : 237-240
                Affiliations
                [1 ]Department of Chemistry, Northwestern University, Evanston, Illinois, USA
                [3 ]Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois, USA
                [4 ]Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA
                [5 ]Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA
                [6 ]Brazilian Center for Protein Research, University of Brasilia, Brasilia, Federal District, Brazil
                [7 ]Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, USA
                [8 ]Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany
                Author notes
                [9 ]Corresponding author: n-kelleher@ 123456northwestern.edu
                [2]

                These authors contributed equally to this work

                Article
                NIHMS746271
                10.1038/nmeth.3731
                4767540
                26780093
                c2f64473-5d31-4133-8bba-09f6c2e918a4

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                Life sciences

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