Reference - Detail
| RRC ID | 15388 |
|---|---|
| Author | Rees JS, Lowe N, Armean IM, Roote J, Johnson G, Drummond E, Spriggs H, Ryder E, Russell S, St Johnston D, Lilley KS. |
| Title | In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry. |
| Journal | Mol Cell Proteomics |
| 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. |
| Volume | 10(6) |
| Pages | M110.002386 |
| Published | 2011-6-1 |
| DOI | 10.1074/mcp.M110.002386 |
| PII | S1535-9476(20)30208-5 |
| PMID | 21447707 |
| PMC | PMC3108830 |
| MeSH | Animals Chromatography, Affinity Drosophila Proteins / chemistry Drosophila Proteins / isolation & purification Drosophila Proteins / metabolism* Drosophila melanogaster / metabolism* Larva / metabolism* Protein Binding Protein Interaction Mapping Protein Phosphatase 1 / chemistry Protein Phosphatase 1 / metabolism Proteome / chemistry Proteome / isolation & purification Proteome / metabolism* Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / isolation & purification Recombinant Fusion Proteins / metabolism* Reproducibility of Results Tandem Mass Spectrometry |
| IF | 4.87 |
| Times Cited | 54 |
| Resource | |
| Drosophila | Fly-trap lines or CPTI lines (Cambridge Protein Trap Insertions lines) |