title: Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors.
abstract: Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks.
authors.affiliation: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Paoletti
authors.forename: Tari J
authors.initials: TJ
authors.lastname: Parmely
authors.forename: Chieri
authors.initials: C
authors.lastname: Tomomori-Sato
authors.forename: Shigeo
authors.initials: S
authors.lastname: Sato
authors.forename: Dongxiao
authors.initials: D
authors.lastname: Zhu
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Conaway
authors.forename: Joan Weliky
authors.initials: JW
authors.lastname: Conaway
authors.forename: Laurence
authors.initials: L
authors.lastname: Florens
authors.forename: Michael P
authors.initials: MP
authors.lastname: Washburn
mesh_terms: D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012319:RNA Polymerase II / Q000378:metabolism*
chemical_list: D011494:Protein Kinases; D012319:RNA Polymerase II