Protocol abstract
Nature Protocols 1, - 1778 - 1789 (2006)
Published online: 16 November 2006 | doi:10.1038/nprot.2006.254
Subject Categories: Biochemistry and protein analysis | Genomics and proteomics | Isolation, purification and separation | Plant biology | Spectroscopy and structural analysis
Quantitative proteomic approach to study subcellular localization of membrane proteins
Pawel G Sadowski1, Tom PJ Dunkley1, Ian P Shadforth2, Paul Dupree3, Conrad Bessant2, Julian L Griffin3 & Kathryn S Lilley1
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.
- Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK.
- Department of Analytical Science and Informatics, Cranfield University, Silsoe MK45 4DT, UK.
- Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK.
Correspondence to: Kathryn S Lilley1 e-mail: k.s.lilley@bioc.cam.ac.uk
