Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors

Andrew C. Paoletti, Tari J. Parmely, Chieri Tomomori-Sato, Shigeo Sato, Dongxiao Zhu, Ronald C. Conaway, Joan Weliky Conaway, Laurence Florens, Michael P. Washburn

Research output: Contribution to journalArticlepeer-review

384 Scopus citations

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 affinitypurified 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.

Original languageEnglish (US)
Pages (from-to)18928-18933
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number50
DOIs
StatePublished - Dec 12 2006
Externally publishedYes

Keywords

  • Mass spectrometry
  • Multidimensional protein identification technology
  • Proteomics
  • Spectrum counting

ASJC Scopus subject areas

  • General

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