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In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.

Rees JS, Lowe N, Armean IM, Roote J, Johnson G, Drummond E, Spriggs H, Ryder E, Russell S, St Johnston D, Lilley KS - Mol. Cell Proteomics (2011)

Bottom Line: This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence.We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets.The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK.

ABSTRACT
Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.

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Interaction network map for FLW, a protein phosphatase that targets Mbs. All putative direct bait-prey interacting proteins immediately surround the bait, FLW, indicated in black. Additional direct interactions from within the entire list are mapped if linked to the bait. Direct ortholog interactions are represented by lighter lines. Indirect interactions have been omitted for simplicity apart from where the “via” proteins are also direct. The size of the circle is proportional to the confidence score of the interaction.
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Figure 5: Interaction network map for FLW, a protein phosphatase that targets Mbs. All putative direct bait-prey interacting proteins immediately surround the bait, FLW, indicated in black. Additional direct interactions from within the entire list are mapped if linked to the bait. Direct ortholog interactions are represented by lighter lines. Indirect interactions have been omitted for simplicity apart from where the “via” proteins are also direct. The size of the circle is proportional to the confidence score of the interaction.

Mentions: Published network maps of our selected proteins are limited and where available, show little overlap depending on the screens, most of which are Y2H. Based on our stringent methodology and validation we have attempted to generate more comprehensive networks to map the proteins we have identified. Fig. 5 shows an interaction network for FLW indicating all putative binary interactions for bait and prey and their binary interactions with other prey in the list (bait-[prey-preyn]n), using full interaction lists that include lower confidence data. Because of the multiple queries in FlyMine (see Methods) we are able to link many more proteins than those that are highlighted on the interaction lists (supplemental Table S4a) as these static lists can show only proteins involved in putative bait-prey or prey-prey interactions and don't distinguish individual pairs, or hubs or any dynamic information. In addition, proteins we found that have interactions in orthologues have also been mapped. For simplicity, indirect interactions have been excluded but are shown in supplemental Table S4a.


In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.

Rees JS, Lowe N, Armean IM, Roote J, Johnson G, Drummond E, Spriggs H, Ryder E, Russell S, St Johnston D, Lilley KS - Mol. Cell Proteomics (2011)

Interaction network map for FLW, a protein phosphatase that targets Mbs. All putative direct bait-prey interacting proteins immediately surround the bait, FLW, indicated in black. Additional direct interactions from within the entire list are mapped if linked to the bait. Direct ortholog interactions are represented by lighter lines. Indirect interactions have been omitted for simplicity apart from where the “via” proteins are also direct. The size of the circle is proportional to the confidence score of the interaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108830&req=5

Figure 5: Interaction network map for FLW, a protein phosphatase that targets Mbs. All putative direct bait-prey interacting proteins immediately surround the bait, FLW, indicated in black. Additional direct interactions from within the entire list are mapped if linked to the bait. Direct ortholog interactions are represented by lighter lines. Indirect interactions have been omitted for simplicity apart from where the “via” proteins are also direct. The size of the circle is proportional to the confidence score of the interaction.
Mentions: Published network maps of our selected proteins are limited and where available, show little overlap depending on the screens, most of which are Y2H. Based on our stringent methodology and validation we have attempted to generate more comprehensive networks to map the proteins we have identified. Fig. 5 shows an interaction network for FLW indicating all putative binary interactions for bait and prey and their binary interactions with other prey in the list (bait-[prey-preyn]n), using full interaction lists that include lower confidence data. Because of the multiple queries in FlyMine (see Methods) we are able to link many more proteins than those that are highlighted on the interaction lists (supplemental Table S4a) as these static lists can show only proteins involved in putative bait-prey or prey-prey interactions and don't distinguish individual pairs, or hubs or any dynamic information. In addition, proteins we found that have interactions in orthologues have also been mapped. For simplicity, indirect interactions have been excluded but are shown in supplemental Table S4a.

Bottom Line: This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence.We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets.The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK.

ABSTRACT
Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.

Show MeSH
Related in: MedlinePlus