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Originally published In Press as doi:10.1074/mcp.T400009-MCP200 on August 4, 2004.
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Molecular & Cellular Proteomics 3:1128-1134, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Localization of Organelle Proteins by Isotope Tagging (LOPIT)*,S

T. P. J. Dunkley{ddagger}, R. Watson§, J. L. Griffin{ddagger}, P. Dupree{ddagger} and K. S. Lilley{ddagger},¶

From the {ddagger} Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom; and § Applied Biosystems, Warrington, Cheshire WA3 7QH, United Kingdom

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.

To whom correspondence should be addressed: Cambridge Centre for Proteomics, Department of Biochemistry, Building O, Downing Site, Cambridge CB2 1QW, United Kingdom. Tel.: 44- (0)1223-765255; Fax: 44-(0)1223-333345; E-mail: k.s.lilley{at}bioc.cam.ac.uk


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