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Mol Biol Cell. 2014 Oct 15;25(20):3178-94. doi: 10.1091/mbc.E14-07-1198. Epub 2014 Aug 27.

Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

Author information

1
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany borner@biochem.mpg.de.
2
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
3
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.

Abstract

We developed "fractionation profiling," a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

PMID:
25165137
PMCID:
PMC4196868
DOI:
10.1091/mbc.E14-07-1198
[Indexed for MEDLINE]
Free PMC Article

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