Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.
Bottom Line: Functionally associated groups of proteins are revealed through cluster analysis.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.In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.
Affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany firstname.lastname@example.org.Show MeSH
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Mentions: Although Drosophila is a widely used model organism, the composition of its CCVs remains poorly characterized. To test whether fractionation profiling is transferable to other cell systems, we chose to investigate Drosophila S2 cells. We applied fractionation profiling without further optimization (Figure 3A). Clathrin and associated proteins had profiles similar to those in HeLa cells (Figure 3B). For proteomic analysis, we prepared a reference and three subfractions from SILAC-labeled S2 cells, repeated the preparation with reversed labeling, and analyzed the six sample pairs by mass spectrometry. In total, we identified >3000 proteins, of which 1799 were quantified across all six samples. PCA shows that subunits of known protein complexes form clusters, as expected (Figure 3C). Known fly CCV proteins, including clathrin, AP-1, and AP-2, formed a distinct cluster in the periphery of the plot. We then constructed a Predictor for the S2 profiling data (Supplemental Table S3). A search against Drosophila clathrin heavy chain revealed a list of 29 candidate CCV proteins predicted with the highest level of confidence (Table 1). Remarkably, the human homologues of 27 of these are known CCV proteins; most of these proteins have not been characterized in Drosophila. An extended search with lower stringency revealed up to 50 candidate CCV proteins (Supplemental Table S4). To validate some of our predictions, we tagged four candidate coat proteins (Figure 3D). All four showed extensive colocalization with established markers of CCVs. In sum, fractionation profiling was successfully implemented to characterize CCVs from S2 cells. Given the evolutionary distance between humans and Drosophila, it is highly likely that the approach will be applicable to a wide variety of cell types.
Affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany email@example.com.