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Universal sample preparation method for proteome analysis

Abstract

We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.

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Figure 1: Filter-aided sample preparation (FASP) for MS-based proteomic analysis.
Figure 2: FASP-based proteomic analysis of SDS lysates.

References

  1. 1

    Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Anal. Chem. 68, 850–858 (1996).

    CAS  Article  Google Scholar 

  2. 2

    Washburn, M.P., Wolters, D. & Yates, J.R., III Nat. Biotechnol. 19, 242–247 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Blonder, J., Xiao, Z. & Veenstra, T.D. Expert Rev. Proteomics 3, 483–496 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Blonder, J., Conrads, T.P. & Veenstra, T.D. Expert Rev. Proteomics 1, 153–163 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Wu, C.C., MacCoss, M.J., Howell, K.E. & Yates, J.R., III Nat. Biotechnol. 21, 532–538 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Olsen, J.V. et al. Mol. Cell. Proteomics 3, 82–92 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Nielsen, P.A. et al. Mol. Cell. Proteomics 4, 402–408 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Le Bihan, T. et al. J. Proteome Res. 5, 2701–2710 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Nagaraj, N., Lu, A., Mann, M. & Wisniewski, J.R. J. Proteome Res. 7, 5028–5032 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Olsen, J.V. et al. Mol. Cell. Proteomics 4, 2010–2021 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Rappsilber, J., Ishihama, Y. & Mann, M. Anal. Chem. 75, 663–670 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Cox, J. & Mann, M. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Shi, R. et al. J. Proteome Res. 6, 2963–2972 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Hubner, N.C., Ren, S. & Mann, M. Proteomics 8, 4862–4872 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Graumann, J. et al. Mol. Cell. Proteomics 7, 672–683 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Mense, S.M. et al. Physiol. Genomics 25, 435–449 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Zhuang, Y., Ma, F., Li-Ling, J., Xu, X. & Li, Y. Mol. Biol. Evol. 20, 1978–1985 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Olsen, J.V. & Mann, M. Proc. Natl. Acad. Sci. USA 101, 13417–13422 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Electrophoresis 20, 3551–3567 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Zubarev, R. & Mann, M. Mol. Cell. Proteomics 6, 377–381 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S. Ren for bioinformatic support. This work was supported by the Max Planck Society for the Advancement of Science, High-throughput Epigenetic Regulatory Organization In Chromatin (HEROIC), Role of Ubiquitin and Ubiquitin-like Modifiers in Cellular Regulation (RUBICON) 6th Framework grants of the European Commission and the Munich Center for Integrated Protein Science (CIPSM).

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Correspondence to Jacek R Wiśniewski or Matthias Mann.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–4, Supplementary Protocol (PDF 1745 kb)

Supplementary Data 1

Analysis of total lysates of Hela cells (XLS 6405 kb)

Supplementary Data 2

Analysis of total lysates of Hela cells fractionated by IEF. (XLS 8640 kb)

Supplementary Data 3

Analysis of total lysates of a mouse brain. (XLS 2253 kb)

Supplementary Data 4

Analysis of total lysate of mouse liver. (XLS 3026 kb)

Supplementary Data 5

Analysis of total lysates of Hela cells digested with different endoproteinases. (XLS 10876 kb)

Supplementary Data 6

Analysis of total lysates of Hela cells digested under different conditions. (XLS 6731 kb)

Supplementary Data 7

Analysis of total lysates of mitochondria isolated from mouse liver. (XLS 236 kb)

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Wiśniewski, J., Zougman, A., Nagaraj, N. et al. Universal sample preparation method for proteome analysis. Nat Methods 6, 359–362 (2009). https://doi.org/10.1038/nmeth.1322

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