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

Nature Methods volume 11, pages 319–324 (2014)Cite this article

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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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Figure 1: Validation of improvements incorporated in the iST method.
Figure 2: Minimal sample-processing protocol performed in an enclosed volume is amenable to automation and scaling.
Figure 3: Quantitative reproducibility of in-depth analysis of S. cerevisiae proteome and copy-number estimation.
Figure 4: In-depth analysis of yeast proteomes and estimation of yeast copy numbers.
Figure 5: In-depth analysis of the human proteome and estimation of copy numbers using the iST method.

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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).

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Authors and 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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  1. Nils A Kulak
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  2. Garwin Pichler
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  4. Nagarjuna Nagaraj
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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.

Corresponding author

Correspondence to Matthias Mann.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Note (PDF 1672 kb)

Supplementary Table 1

Protein identifications and protein copy number estimations of S.cerevisiae, S.pombe and HeLa cells. (XLSX 1707 kb)

Supplementary Table 2

Orthologs of S.cerevisiae, S.pombe and HeLa cells and their protein copy number estimations. (XLSX 931 kb)

Supplementary Table 3

Buffer compositions and materials for the iST method. (XLSX 12 kb)

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. (MP4 27589 kb)

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Kulak, N., Pichler, G., Paron, I. et al. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319–324 (2014). https://doi.org/10.1038/nmeth.2834

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