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Compartmentalization of the eukaryotic cell into organelles and protein complexes is fundamental in creating controlled microenvironments for diverse biological functions. Location of proteins within specific intracellular niches has an influence on their activity, stability, post-translational modifications, and ability to interact with other protein partners. Knowledge of protein sub-cellular localization is of paramount importance in the analysis of protein function. The majority of sub-cellular compartments cannot be purified to homogeneity making assignment of their genuine residents problematic. We have developed LOPIT (localization of organelle proteins by isotope tagging), a high-throughput proteomics based technique for protein localization to sub-cellular organelles a. This approach makes use of the fact that organelles and larger protein complexes have specific distribution profiles within density gradients. Organelle and complex specific distribution patterns can be assigned by determining the distribution of known marker proteins using differential stable isotope labeling. Distribution patterns of Ħunknown˘ proteins can then be revealed using a variety of statistical tools to match these to organelle/complex specific patterns. We have applied the LOPIT technique to study sub-cellular location of proteins in a number of systems including plant cells, vertebrate cell culture and whole organisms b,c,d. A review of the LOPIT technique will be given along with a description of how we are extending it to dynamically map protein redistribution upon a given perturbation. Furthermore, the importance of interrogating data structure and carrying out suitable number of replicate experiments to overcome biological and technical variance in large scale quantitative approaches such as LOPIT will be discussed. a. Sadowski, PG Dunkley, TPJ, Shadforth, IP,Griffin, JL, Dupree, P, Bessant, C, Lilley, KS, (2006) Quantitative proteomic approach to study subcellular localization of membrane proteins. Nature Protocols 1; 1778 - 1789 b. Dunkley TP, Hester S, Shadforth IP, Runions J, Weimar T, Hanton SL, Griffin JL, Bessant C, Brandizzi F, Hawes C, Watson RB, Dupree P, Lilley KS. (2006) Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci 103(17):6518-23. c. Hall SL, Hester S, Griffin JL, Lilley KS, Jackson AP (2009) The organelle proteome of the DT40 Lymphocyte cell line. Mol Cell Proteomics. 8(6):1295-305 d. Tan D, Dvinge H, Christoforou A, Bertone P, Martinez Arias A, Lilley KS. (2009) Mapping organelle proteins and protein complexes in Drosophila melanogaster. J Proteome Res 8(6):2667-2678 |
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