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Quantitative proteomic approach to study subcellular localization of membrane proteins

Nature Protocols volume 1, pages 1778–1789 (2006)Cite this article

Abstract

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.

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Figure 1: General principles of the LOPIT approach.
Figure 2: Structure of the iTRAQ reagent.
Figure 3: Overview of the cell fractionation procedure.
Figure 4: Western blotting analysis.
Figure 5: Peptide fragmentation by CID.
Figure 6: PCA analysis showing clustering of proteins according to their distribution down the gradient.

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Authors and Affiliations

  1. Department of Biochemistry, Cambridge Centre for Proteomics, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK

    Pawel G Sadowski, Tom PJ Dunkley & Kathryn S Lilley

  2. Department of Analytical Science and Informatics, Cranfield University, Silsoe, MK45 4DT, UK

    Ian P Shadforth & Conrad Bessant

  3. Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK

    Paul Dupree & Julian L Griffin

Authors
  1. Pawel G Sadowski
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  2. Tom PJ Dunkley
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  3. Ian P Shadforth
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  4. Paul Dupree
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  6. Julian L Griffin
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  7. Kathryn S Lilley
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Correspondence to Kathryn S Lilley.

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Sadowski, P., Dunkley, T., Shadforth, I. et al. Quantitative proteomic approach to study subcellular localization of membrane proteins. Nat Protoc 1, 1778–1789 (2006). https://doi.org/10.1038/nprot.2006.254

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