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A method for the comprehensive proteomic analysis of membrane proteins

Nature Biotechnology volume 21, pages 532–538 (2003)Cite this article

Abstract

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.

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Figure 1: Application of the hpPK method to complex membrane-containing samples.
Figure 2: Characterization of membrane protein topology and relative protein localization.
Figure 3: Distribution of transmembrane domains and total sequence coverage in identified brain proteins.
Figure 4: Comprehensive characterization of individual proteins from unfractionated rat brain homogenates.
Figure 5: Comprehensive characterization of a Golgi membrane protein from a global protease protection analysis.

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Acknowledgements

The authors gratefully acknowledge financial support from the American Cancer Society PF-03-065-01-MGO (C.C.W.) and the National Institute of Health grants F32DK59731 (M.J.M.), RO1-GM42629 (K.E.H) and R33 CA81665 and RR11823 (J.R.Y.).

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Author notes

  1. Christine C. Wu and Michael J. MacCoss: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, The Scripps Research Institute, La Jolla, 92037, CA

    Christine C. Wu, Michael J. MacCoss & John R. Yates III

  2. Department of Cellular and Structural Biology University of Colorado Health Sciences Center, University of Colorado Health Sciences Center, Denver, 80206, CO

    Kathryn E. Howell

  3. Department of Proteomics and Metabolomics, Torrey Mesa Research Institute, 3115 Merryfield Row, San Diego, 92121-1125, CA

    John R. Yates III

Authors
  1. Christine C. Wu
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  4. John R. Yates III
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Correspondence to John R. Yates III.

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Wu, C., MacCoss, M., Howell, K. et al. A method for the comprehensive proteomic analysis of membrane proteins. Nat Biotechnol 21, 532–538 (2003). https://doi.org/10.1038/nbt819

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