Article | Published:

Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells

Nature Methods volume 11, pages 319324 (2014) | Download Citation

Abstract

Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R2 = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R2 = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

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Acknowledgements

We thank S. Braun and P. Georgescu (Ludwig Maximilian University of Munich) for providing us with fission yeast pellets, F. Sacco, M. Steger, D. Walther and M. Räschle for help concerning biological interpretations. M. Hein, A. Dalfovo, C. Schaab and J. Cox helped with figures, videos and bioinformatics analysis of our data sets. Work in M.M.'s laboratory is supported by the Max Planck Society for the Advancement of Science and PROSPECT, a 7th framework program of the European Union (grant agreement HEALTH-F4-2008-201648).

Author information

Affiliations

  1. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

    • Nils A Kulak
    • , Garwin Pichler
    • , Igor Paron
    • , Nagarjuna Nagaraj
    •  & Matthias Mann

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Contributions

N.A.K. and M.M. developed and invented the method; G.P. and N.N. contributed in the developments; N.A.K., G.P., I.P. and N.N. performed the experiments; and N.A.K., G.P., N.N. and M.M. designed and interpreted the experiments, and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthias Mann.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11 and Supplementary Note

Excel files

  1. 1.

    Supplementary Table 1

    Protein identifications and protein copy number estimations of S.cerevisiae, S.pombe and HeLa cells.

  2. 2.

    Supplementary Table 2

    Orthologs of S.cerevisiae, S.pombe and HeLa cells and their protein copy number estimations.

  3. 3.

    Supplementary Table 3

    Buffer compositions and materials for the iST method.

Videos

  1. 1.

    iST sample-preparation video tutorial.

    The video tutorial shows all steps of the iST sample preparation workflow. It also shows how to troubleshoot the method, if necessary.

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DOI

https://doi.org/10.1038/nmeth.2834

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