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Determining proteome-wide expression levels using reverse protein arrays in fission yeast

Nature Protocols volume 7, pages 1830–1835 (2012)Cite this article

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Abstract

Global protein expression profiling of various mutants or growth conditions is currently a major challenge in biology. Here we provide a protocol for a strategy that we recently developed that couples ORFeome-based (ORF denotes open reading frame) expression to reverse protein arrays; this approach accurately quantifies more than 99% of the predicted fission yeast proteins in various genetic backgrounds. The first stage of this two-stage protocol requires mass mating between any fertile fission yeast mutant of interest and the integrated fission yeast–tagged ORFeome followed by selection of recombinant haploids. The second stage of the protocol, called reverse protein arrays, involves simple large-scale extraction of total proteins, which are then spotted on nitrocellulose membranes for detection by quantitative dot blot. When handled manually, the entire protocol takes about 2 months. However, the process could easily be automated and should also be applicable to other organisms.

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Figure 1: Schematic workflow of the PROCEDURE.

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References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    CAS  Google Scholar 

  2. Picotti, P., Bodenmiller, B., Mueller, L.N., Domon, B. & Aebersold, R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138, 795–806 (2009).

    Article  CAS  Google Scholar 

  3. Uhlen, M. Affinity as a tool in life science. Biotechniques 44, 649–654 (2008).

    Article  CAS  Google Scholar 

  4. Uhlen, M., Graslund, S. & Sundstrom, M. A pilot project to generate affinity reagents to human proteins. Nat. Methods 5, 854–855 (2008).

    Article  CAS  Google Scholar 

  5. Colwill, K. & Graslund, S. A roadmap to generate renewable protein binders to the human proteome. Nat. Methods 8, 551–558 (2011).

    Article  CAS  Google Scholar 

  6. Matsuyama, A. et al. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol. 24, 841–847 (2006).

    Article  CAS  Google Scholar 

  7. Kim, D.U. et al. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol. 28, 617–623 (2010).

    Article  CAS  Google Scholar 

  8. Spirek, M. et al. S. pombe genome deletion project: an update. Cell Cycle 9, 2399–2402 (2010).

    Article  CAS  Google Scholar 

  9. Bauer, F. et al. Translational control of cell division by Elongator. Cell Rep. 1, 424–433 (2012).

    Article  CAS  Google Scholar 

  10. Dewez, M. et al. The conserved Wobble uridine tRNA thiolase Ctu1-Ctu2 is required to maintain genome integrity. Proc. Natl. Acad. Sci. USA 105, 5459–5464 (2008).

    Article  CAS  Google Scholar 

  11. Nugent, R.L. et al. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation. BMC Genomics 11, 59 (2010).

    Article  Google Scholar 

  12. Baryshnikova, A. et al. Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Methods Enzymol. 470, 145–179 (2010).

    Article  CAS  Google Scholar 

  13. Coluccio, A.E., Rodriguez, R.K., Kernan, M.J. & Neiman, A.M. The yeast spore wall enables spores to survive passage through the digestive tract of Drosophila. PLoS ONE 3, e2873 (2008).

    Article  Google Scholar 

  14. Maundrell, K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J. Biol. Chem. 265, 10857–10864 (1990).

    Article  CAS  Google Scholar 

  15. Bamps, S. et al. Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast. Biochem. Biophys. Res. Commun. 325, 1424–1432 (2004).

    Article  CAS  Google Scholar 

  16. Fersht, N., Hermand, D. & Nurse, P. Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast. Nucleic Acids Res. 35, 5323–5337 (2007).

    Article  CAS  Google Scholar 

  17. Coudreuse, D. et al. A gene-specific requirement of RNA polymerase II CTD phosphorylation for sexual differentiation in S. pombe. Curr. Biol. 20, 1053–1064 (2010).

    Article  CAS  Google Scholar 

  18. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the GEMO laboratory for discussions. This work was supported by a European Molecular Biology Organization (EMBO) short-term fellowship (58-2008), and grants FRFC 2.4510.10, Credit aux chercheurs 1.5.013.09 and MIS F.4523.11 to D.H.; F.B. is a Fonds de la Recherche en Industrie et Agriculture (FRIA) Research Fellow; D.H. is a Fonds National de la Recherche Scientifique (FNRS; Belgium) Research Associate.

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Authors and Affiliations

  1. Namur Research College, The University of Namur, Namur, Belgium

    Fanelie Bauer & Damien Hermand

  2. Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan

    Fanelie Bauer, Akihisa Matsuyama & Minoru Yoshida

Authors
  1. Fanelie Bauer
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  2. Akihisa Matsuyama
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  3. Minoru Yoshida
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  4. Damien Hermand
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Contributions

F.B. designed and performed the experiments, and wrote the manuscript; A.M. and M.Y. designed the reverse protein array strategy, and helped with its development; D.H. designed the study and wrote the manuscript.

Corresponding author

Correspondence to Damien Hermand.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Comparison of α-tubulin levels between the wild type and the mutant strains. Total extracts of biological triplicates of wild type and mutant (Δelp3Δctu1) strains have been collected and separated by PAGE; transferred onto nitrocellulose membrane, incubated with anti-α–tubulin primary antibodies, secondary fluorescent antibodies and scanned to measure the fluorescent signal of each lane. The resulting raw and mean values are indicated. (PDF 319 kb)

Supplementary Figure 2

A representative membrane after hybridization and scanning. Total extracts of wild type ORFeome were spotted in quadruplicates onto nitrocellulose membrane, incubated with anti-α–tubulin and anti-His6 primary antibodies, secondary fluorescent antibodies and scanned to measure the fluorescent signal of each dot. As expected, the level of α-tubulin does not show strong variation from one strain to another while tagged ORFeome proteins display a much broader range of expression level. (PDF 1452 kb)

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Bauer, F., Matsuyama, A., Yoshida, M. et al. Determining proteome-wide expression levels using reverse protein arrays in fission yeast. Nat Protoc 7, 1830–1835 (2012). https://doi.org/10.1038/nprot.2012.114

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