Quantitative Proteomic Analysis of Distinct Mammalian Mediator Complexes Using Normalized Spectral Abundance Factors

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2006-12, Vol.103 (50), p.18928-18933
Main Authors: Paoletti, Andrew C., Parmely, Tari J., Tomomori-Sato, Chieri, Sato, Shigeo, Zhu, Dongxiao, Conaway, Ronald C., Weliky Conaway, Joan, Florens, Laurence, Washburn, Michael P.
Format: Article
Language:English
Subjects:
Biochemistry
Biological Sciences
Cyclins
Datasets
Health care industry
HeLa Cells
Humans
Mass spectrometry
Mass spectroscopy
Medical research
Models, Biological
Protein Kinases - metabolism
Proteins
Proteomics
Proteomics - methods
Ribonucleic acid
RNA
RNA Polymerase II - metabolism
Spectroscopic analysis
Western blotting
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0606379103