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High-throughput generation of selected reaction-monitoring assays for proteins and proteomes

Nature Methods volume 7, pages 4346 (2010) | Download Citation

Abstract

Selected reaction monitoring (SRM) uses sensitive and specific mass spectrometric assays to measure target analytes across multiple samples, but it has not been broadly applied in proteomics owing to the tedious assay development process for each protein. We describe a method based on crude synthetic peptide libraries for the high-throughput development of SRM assays. We illustrate the power of the approach by generating and applying validated SRM assays for all Saccharomyces cerevisiae kinases and phosphatases.

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Acknowledgements

We acknowledge A. Wepf for assistance with the shotgun measurements on an Orbitrap instrument. We thank R. Volkmer, T. Meyer and M. Teruel for insightful discussions. This project was funded in part by ETH Zurich, the Swiss National Science Foundation, US National Heart, Lung, and Blood Institute, National Institutes of Health (N01-HV-28179) and by SystemsX.ch, the Swiss initiative for systems biology. F.D. was supported by the Helmholtz Alliance on Systems Biology. P.P. is the recipient of a Marie Curie Intra-European fellowship.

Author information

Affiliations

  1. Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Zurich, Switzerland.

    • Paola Picotti
    • , Oliver Rinner
    • , Robert Stallmach
    • , Bruno Domon
    •  & Ruedi Aebersold
  2. Biognosys AG, Zurich, Switzerland.

    • Oliver Rinner
  3. Department of Proteomics, Helmholtz Centre for Environmental Research, Leipzig, Germany.

    • Franziska Dautel
  4. Institute for Systems Biology, Seattle, Washington, USA.

    • Terry Farrah
    •  & Ruedi Aebersold
  5. JPT Peptide Technologies GmbH, Berlin, Germany.

    • Holger Wenschuh
  6. Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland.

    • Ruedi Aebersold
  7. Faculty of Science, University of Zurich, Zurich, Switzerland.

    • Ruedi Aebersold

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Contributions

P.P. and R.A. designed the experiments and the method workflow and wrote the paper. O.R. conceived the data analysis pipeline and performed part of the data analysis. P.P., R.S. and F.D. performed the experiments and analyzed data. T.F. compared results with those of ref. 15. H.W. supervised optimization of the SPOT-synthesis technology for producing small-scale, unpurified peptides to be used in SRM or multiple reaction monitoring assays; B.D. contributed to technical aspects of the SRM or MRM approach development. R.A. supervised the project.

Competing interests

A patent application protecting the technology described in this paper has been filed by P.P., R.A. and O.R. (EP 08 009454.3, PCT/EP09/03611). O.R. is affiliated with Biognosys AG (Zurich, Switzerland). H.W. is an employee of JPT Peptide Technologies GmbH (Berlin, Germany).

Corresponding author

Correspondence to Ruedi Aebersold.

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    Supplementary Figures 1–7, Supplementary Tables 1–7 and Supplementary Discussion

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DOI

https://doi.org/10.1038/nmeth.1408

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