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The study of macromolecular complexes by quantitative proteomics

Abstract

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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Figure 1: Schematic representation of the quantitative proteomics approach for the analysis of affinity purified macromolecular complexes.
Figure 2: Single-step promoter DNA affinity purification of an RNA Pol II preinitiation complex from nuclear extracts.
Figure 3: Use of relative quantification to guide identification of specific complex components in complex mixtures.

References

  1. 1

    Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    CAS  Article  Google Scholar 

  2. 2

    Ranish, J.A., Yudkovsky, N. & Hahn, S. Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13, 49–63 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Hartwell, L.H., Hopfield, J.J., Leibler, S. & Murray, A.W. From molecular to modular cell biology. Nature 402, C47–C52 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Fields, S. & Song, O.-k. A novel genetic system to detect protein–protein interactions. Nature 340, 245–246 (1989).

    CAS  Article  Google Scholar 

  5. 5

    Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    CAS  Article  Google Scholar 

  7. 7

    Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Zhou, H., Ranish, J.A., Watts, J.D. & Aebersold, R. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat. Biotechnol. 20, 512–515 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Cagney, G. & Emili, A. De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging. Nat. Biotechnol. 20, 163–170 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Han, D.K., Eng, J., Zhou, H. & Aebersold, R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946–951 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Eng, J.K., McCormack, A.L. & Yates, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in protein databases. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    CAS  Article  Google Scholar 

  12. 12

    Lee, T.I. & Young, R.A. Transcription of eukaryotic protein-coding genes. Annu. Rev. Genet. 34, 77–137 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Li, X.Y., Bhaumik, S.R. & Green, M.R. Distinct classes of yeast promoters revealed by differential TAF recruitment. Science 288, 1242–1244 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Kuras, L., Kosa, P., Mencia, M. & Struhl, K. TAF-containing and TAF-independent forms of transcriptionally active TBP in vivo. Science 288, 1244–1248 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Fry, C.J. & Peterson, C.L. Chromatin remodeling enzymes: who's on first? Curr. Biol. 11, R185–R197 (2001).

    CAS  Article  Google Scholar 

  16. 16

    Reddy, P. & Hahn, S. Dominant negative mutations in yeast TFIID define a bipartite DNA-binding region. Cell 65, 349–357 (1991).

    CAS  Article  Google Scholar 

  17. 17

    Costanzo, M.C. et al. YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information. Nucleic Acids Res. 29, 75–79 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Zhang, X., Jin, Q.K., Carr, S.A. & Annan, R.S. N-terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry. Rapid Commun. Mass Spectrom. 16, 2325–2332 (2002).

    CAS  Article  Google Scholar 

  19. 19

    Yudkovsky, N., Ranish, J.A. & Hahn, S. A transcription reinitiation intermediate that is stabilized by activator. Nature 408, 225–229 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Auble, D.T. et al. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8, 1920–1934 (1994).

    CAS  Article  Google Scholar 

  21. 21

    Sprague, G.F. & Thorner, J.W. Pherormone response and signal transduction during the mating process of Saccharomyces cerevisiae. in The Molecular and Cellular Biology of the Yeast Saccharomyces Vol. 2 (eds. Jones, E.W., Pringle, J.R. & Broach, J.R.) 657–744 (Cold Spring Harbor Laboratory Press, Plainview, 1992).

    Google Scholar 

  22. 22

    Tedford, K., Kim, S., Sa, D., Stevens, K. & Tyers, M. Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates. Curr. Biol. 7, 228–238 (1997).

    CAS  Article  Google Scholar 

  23. 23

    Olson, M.V. Genome structure and organization in Saccharomyces cerevisiae. in The Molecular and Cellular Biology of the Yeast Saccharomyces Vol. 1 (eds. Broach, J.R., Pringle, J. & Jones, E.) 1–39 (Cold Spring Harbor Laboratory Press, Plainview, 1991).

    Google Scholar 

  24. 24

    Roberts, C.J. et al. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287, 873–880 (2000).

    CAS  Article  Google Scholar 

  25. 25

    Griffin, T.J. et al. Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer. Anal. Chem. 73, 978–986 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Griffin, T.J. et al. Toward a high-throughput approach to quantitative proteomic analysis: expression-dependent protein identification by mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 1238–1246 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Shiio, Y. et al. Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21, 5088–5096 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Kang, J.J., Auble, D.T., Ranish, J.A. & Hahn, S. Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription. Mol. Cell. Biol. 15, 1234–1243 (1995).

    CAS  Article  Google Scholar 

  29. 29

    Aitchison, J.D., Rout, M.P., Marelli, M., Blobel, G. & Wozniak, R.W. Two novel related yeast nucleoporins Nup170p and Nup157p: complementation with the vertebrate homologue Nup155p and functional interactions with the yeast nuclear pore-membrane protein Pom152p. J. Cell Biol. 131, 1133–1148 (1995).

    CAS  Article  Google Scholar 

  30. 30

    Yi, E.C. et al. Approaching complete peroxisome characterization by gas-phase fractionation. Electrophoresis 23, 3205–3216 (2002).

    CAS  Article  Google Scholar 

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Acknowledgements

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.

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Correspondence to Ruedi Aebersold.

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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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