Direct analysis of protein complexes using mass spectrometry

Abstract

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Direct analysis of large protein complexes (DALPC).
Figure 2: Identifying proteins in the yeast ribosome complex using DALPC.
Figure 3: Identifying ribosomal proteins in a total yeast extract using DALPC.
Figure 4: Integrated DALPC.
Figure 5: Localizing YMR116p to the yeast 40S ribosomal subunit.
Figure 6: Localizing RACK1 to the human 40S ribosomal subunit.

References

  1. 1

    Pugh, B.F. Mechanisms of transcription complex assembly. Curr. Opin. Cell Biol. 8, 303–311 ( 1996).

    CAS  Article  Google Scholar 

  2. 2

    Wool, I.G., Chan, Y.-L. & Gluck, A. Structure and evolution of mammalian ribosomal proteins. Biochem. Cell Biol. 73, 933– 947 (1995).

    CAS  Article  Google Scholar 

  3. 3

    Mager, W.H. et al. A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Nucleic. Acids Res. 25, 4872–4875 (1997).

    CAS  Article  Google Scholar 

  4. 4

    Phizicky, E.M. & Fields, S. Protein–protein interactions: methods for detection and analysis. Microbiol. Rev. 59, 94–123 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Neubauer, G. et al. Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. Proc. Natl. Acad. Sci. USA 94, 385–390 ( 1997).

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Link, A.J., Carmack, E. & Yates, J.R. A strategy for the identification of proteins localized to subcellular spaces: applications to E. coli periplasmic proteins. Int. J. Mass Spectrom. Ion Proc. 160, 303 –316 (1997).

    CAS  Article  Google Scholar 

  8. 8

    McCormack, A.L. et al. Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767–776 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Giddings, J.C. Concepts and comparisons in multidimensional separation. J. High Resol. Chromatogr. Commun. 10, 319– 323 (1987).

    CAS  Article  Google Scholar 

  10. 10

    Lundell, N. & Markides, K. Two-dimensional liquid chromatography of peptides: an optimization of strategy. Chromatographia 34, 369–375 (1992).

    CAS  Article  Google Scholar 

  11. 11

    O'Farrell, P.H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Takahashi, N., Ishioka, N., Takahashi, Y. & Putnam, F.W. Automated tandem high-performance liquid chromatographic system for separation of extremely complex peptide mixtures. J. Chromatogr. 326, 407-418 (1985).

    CAS  Article  Google Scholar 

  13. 13

    Opiteck, G.J., Lewis, K.C. & Jorgenson, J.W. Comprehensive on-line LC/LC/MS of proteins. Anal. Chem. 69, 1518–1524 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Yates, J.R., Eng, J.K., McCormack, A.L. & Schieltz, D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426–1436 (1995).

    CAS  Article  Google Scholar 

  15. 15

    Otaka, E. & Osawa, S. Yeast ribosomal proteins: V. Correlation of several nomenclatures and proposal of a standard nomenclature. Mol. Gen. Genet. 181, 176–182 (1981).

    CAS  Article  Google Scholar 

  16. 16

    Mager, W.H. & Planta, R.J. Coordinate expression of ribosomal protein genes in yeast as a function of cellular growth rate. Mol. Cell. Biol. 104, 181–187 (1991).

    CAS  Google Scholar 

  17. 17

    Warner, J. Synthesis of ribosomes in Saccharomyces cerevisiae. Microbiol. Rev. 53, 256–271 ( 1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Link, A.J., Hays, L.G., Carmack, E.B. & Yates, J.R. Identifying the major proteome components of Haemophilus influenzae type-strain NCTC 8143. Electrophoresis 18, 1314–1334 (1997).

    CAS  Article  Google Scholar 

  19. 19

    Gatlin, C.L., Kleemann, G.R., Hays, L.G., Link, A.J. & Yates, J.R. Protein identification at the low femtomole level from silver stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem. 263, 93– 101 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Cherry, J.M. et al. Genetic and physical maps of Saccharomyces cerevisiae. Nature 387, 67–73 (1997).

    CAS  Article  Google Scholar 

  21. 21

    Ron, D. et al. Cloning of an intracellular receptor for protein kinase C: a homolog of the β subunit of G proteins. Proc. Natl. Acad. Sci. USA 91, 839–843 ( 1994).

    CAS  Article  Google Scholar 

  22. 22

    Chantrel, Y., Gaisne, M., Lions, C. & Verdiere, J. The transcriptional regulator Hap1p (Cyp1p) is essential for anaerobic or heme-deficient growth of Saccharomyces cerevisiae: genetic and molecular characterization of an extragenic suppressor that encodes a WD repeat protein. Genetics 148, 559–569 ( 1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Battaner, E. & Vazquez, D. Preparation of active 60S and 40S subunits from yeast ribosomes. Methods Enzymol. 20, 446–449 (1971).

    Article  Google Scholar 

  24. 24

    Warner, J.R. & Gorenstein, C. in Methods in cell biology. (ed.Prescott, D.M.) 45–60 (Academic, New York; 1978).

    Google Scholar 

  25. 25

    Jones, E.W. Tackling the protease problem in Saccharomyces cerevisiae. Methods. Enzymol. 194, 428–453 (1991).

    CAS  Article  Google Scholar 

  26. 26

    Raue, H.A., Mager, W.H. & Planta, R.J. Structural and functional analysis of yeast ribosomal proteins. Methods Enzymol. 194, 453– 477 (1991).

    CAS  Article  Google Scholar 

  27. 27

    Wessel, D. & Flügge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141– 143 (1984).

    CAS  Article  Google Scholar 

  28. 28

    Drubin, D.G., Miller, K.G. & Botstein, D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J. Cell Biol. 107, 2551– 2561 (1988).

    CAS  Article  Google Scholar 

  29. 29

    Ruan, H., Brown, C.Y. & Morris, D.R. in mRNA formation and function. (ed. Richter, J.D.) 305–321 (Academic, New York; 1997).

    Google Scholar 

  30. 30

    Saccharomyces genome database at http://genome-www.stanford.edu/Saccharomyces/.

  31. 31

    Güldener, U., Heck, S., Fiedler, T., Beinhauer, J. & Hegemann, J.H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519–2524 (1996).

    Article  Google Scholar 

  32. 32

    Direct Analysis of Large Protein Complexes (DALPC) at http://thompson.mbt.washington.edu/dalpc/.

Download references

Acknowledgements

We thank D. Tabb, L. Hayes, G. Kleeman, E. Malone, and M. Olson for critical reading of the manuscript and T. Gatlin for assistance with the figures. Partially supported by NIH grant CA39053 (G.T.M and D.R.M). Supported by National Center for Research Resources Yeast Center grant RR11823, NIH grant GM52095 and NSF's Science and Technology Center grant BIR 8809710 to J.R.Y., and NIH postdoctoral fellowship grant T32 HG00035-03 to A.J.L.

Author information

Affiliations

Authors

Corresponding author

Correspondence to John R. Yates III.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Link, A., Eng, J., Schieltz, D. et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17, 676–682 (1999). https://doi.org/10.1038/10890

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing