We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded affinity tag (ICAT) reagents1 and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure2. In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.
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We thank S. Hahn for the gift of rTBP and antibodies to TBP, TFIIB and SRB4, A. Nesvizhskii for help with data analysis and J. Aitchison, M. Wright and B. Wollscheid for comments on the manuscript. This work was supported by grants from the US National Cancer Institute and US National Institutes of Health Research Resource Center, by federal funds from the National Heart, Lung, and Blood Institute of the National Institutes of Health and by a postdoctoral fellowship from the National Institutes of Health to J.A.R. Partial funding for this work came through a gift from Merck and Co. to the Institute for Systems Biology.
The authors declare no competing financial interests.
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Ranish, J., Yi, E., Leslie, D. et al. The study of macromolecular complexes by quantitative proteomics. Nat Genet 33, 349–355 (2003). https://doi.org/10.1038/ng1101
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