Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

One-hour proteome analysis in yeast

Abstract

Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-μm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350–375 nl/min) that increases the number of proteins identified across a single liquid chromatography–tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes 24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Structure of the in-house-manufactured column heater.
Figure 2: Column fabrication.
Figure 3: Effect of MS1 AGC target, resolution and MS2 max inject time on the number of MS/MS scans, PSMs and unique peptides.
Figure 4: Yeast peptide and protein identifications for all replicates.
Figure 5: Unique peptides and proteins identified over the LC-MS/MS gradient.
Figure 6: Effect of gradient length on peptide and protein identifications.

References

  1. Tabb, D.L. et al. Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. J. Proteome Res. 9, 761–776 (2010).

    CAS  Article  Google Scholar 

  2. Liu, H., Sadygov, R.G. & Yates, J.R. III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76, 4193–4201 (2004).

    CAS  Article  Google Scholar 

  3. Washburn, M.P., Wolters, D. & Yates, J.R. III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    CAS  Article  Google Scholar 

  4. Hebert, A.S. et al. The one hour yeast proteome. Mol. Cell Proteomics 13, 339–347 (2014).

    CAS  Article  Google Scholar 

  5. Senko, M.W. et al. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Anal. Chem. 85, 11710–11714 (2013).

    CAS  Article  Google Scholar 

  6. Goffeau, A. et al. Life with 6000 genes. Science 274 546 563–547 (1996).

    Article  Google Scholar 

  7. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    CAS  Article  Google Scholar 

  8. de Godoy, L.M. et al. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455, 1251–1254 (2008).

    CAS  Article  Google Scholar 

  9. Wu, R. et al. Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes. Mol. Cell Proteomics 10, M111.009654 (2011).

    Article  Google Scholar 

  10. Webb, K.J., Xu, T., Park, S.K. & Yates, J.R. III. Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast. J. Proteome Res. 12, 2177–2184 (2013).

    CAS  Article  Google Scholar 

  11. Nagaraj, N. et al. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Mol. Cell Proteomics 11, M111.013722 (2012).

    Article  Google Scholar 

  12. Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014).

    CAS  Article  Google Scholar 

  13. Meyer, J.G. & Komives, E.A. Charge state coalescence during electrospray ionization improves peptide identification by tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 23, 1390–1399 (2012).

    CAS  Article  Google Scholar 

  14. Hahne, H. et al. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments. Nat. Methods 10, 989–991 (2013).

    CAS  Article  Google Scholar 

  15. Pirmoradian, M. et al. Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Mol. Cell Proteomics 12, 3330–3338 (2013).

    CAS  Article  Google Scholar 

  16. Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  Google Scholar 

  17. Richards, A., Hebert, A., Ulbrich, A., Bailey, D., Coughlin, E., Westphall, M. & Coon, J. Preparation of yeast cells for proteomic analysis by LC-MS/MS. Protocol Exchange (2015) 10.1038/protex.2015.030.

  18. Treco, D.A. & Winston, F. Growth and manipulation of yeast. Curr. Protoc. Mol. Biol. 82, 13.2.1–13.2.12 (2008).

    Article  Google Scholar 

  19. Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    CAS  Article  Google Scholar 

  20. Wenger, C.D., Phanstiel, D.H., Lee, M.V., Bailey, D.J. & Coon, J.J. COMPASS: a suite of pre- and post-search proteomics software tools for OMSSA. Proteomics 11, 1064–1074 (2011).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to A. Merrill for yeast production. We thank A. Gasch for assistance with yeast growth. This work was supported by the US National Institutes of Health (R01 GM080148) and the National Science Foundation (0701846). A.L.R. gratefully acknowledges the support from a US National Institutes of Health–funded Genomic Sciences Training Program (5T32HG002760).

Author information

Authors and Affiliations

Authors

Contributions

A.L.R. and A.S.H. designed experiments, performed research, analyzed data and wrote the paper; D.J.B. contributed analysis tools, analyzed data and wrote the paper; A.U. and E.E.C. contributed materials; M.S.W. analyzed data; J.J.C. designed the research and wrote the paper.

Corresponding author

Correspondence to Joshua J Coon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Distribution of the intensities of peptide precursors in the survey scan.

Red is with and black is without the addition of DMSO.

Supplementary Figure 2 Location of instrument parameters within Method Editor on the Orbitrap Fusion.

(A) MS1 resolution, MS1 detector type, and MS1 AGC target are set within the MS OT section. (B) Top speed data dependent mode and precursor priority are selected within the Decisions section. (C) MS2 detector type, MS2 isolation window, MS2 AGC target, MS2 max injection time, activation type, collision energy, detector type and scan rate are set within the MS/MS IT section.

Supplementary Figure 3 Orbitrap Fusion scan sequence.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–3 and Supplementary Data (PDF 1727 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Richards, A., Hebert, A., Ulbrich, A. et al. One-hour proteome analysis in yeast. Nat Protoc 10, 701–714 (2015). https://doi.org/10.1038/nprot.2015.040

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.040

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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