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Universal sample preparation method for proteome analysis

Nature Methods volume 6, pages 359–362 (2009)Cite this article

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.

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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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Authors and Affiliations

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

    Jacek R Wiśniewski, Alexandre Zougman, Nagarjuna Nagaraj & Matthias Mann

Authors
  1. Jacek R Wiśniewski
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  2. Alexandre Zougman
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  3. Nagarjuna Nagaraj
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  4. Matthias Mann
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Corresponding authors

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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