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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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Related in: MedlinePlus

Analysis of negative control data. A, Multiple parallel affinity purifications of 25 nontagged lines (negative controls) and the use of ProteinCenter™ identified contaminants specific to FLAG and StrepII resins (FLAGome and STREPome respectively) and those common to both (BEADome). After identifying and removing the single protein hits (subsets) for each affinity method there is 72% overlap. B, The top ten hits for the FLAGome, STREPome and BEADome are listed in the table and the complete listings are presented in supplemental Tables S3A–S3C online. The top ten interacting proteins common to multiple baits are listed as the TRAPome and the complete list is presented in supplemental Table S5 online.
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Figure 4: Analysis of negative control data. A, Multiple parallel affinity purifications of 25 nontagged lines (negative controls) and the use of ProteinCenter™ identified contaminants specific to FLAG and StrepII resins (FLAGome and STREPome respectively) and those common to both (BEADome). After identifying and removing the single protein hits (subsets) for each affinity method there is 72% overlap. B, The top ten hits for the FLAGome, STREPome and BEADome are listed in the table and the complete listings are presented in supplemental Tables S3A–S3C online. The top ten interacting proteins common to multiple baits are listed as the TRAPome and the complete list is presented in supplemental Table S5 online.

Mentions: MASCOT results were imported the into the ProteinCenter™ software package (see methods) to compare the FLAG and StrepII interaction data sets, compare Mascot protein probability scores of proteins identified in controls and test samples, and subtract nonspecific proteins that were pulled down in nontagged lines and no-protein systematic controls (supplemental Fig. S2). The software was also used to compare proteins from multiple pull-downs and highlight contaminants specific to each or both resins. Proteins consistently identified with FLAG resins only (FLAGome), StrepII resins only (STREPome) and those binding to both resins (BEADome) are listed in full (supplemental Tables S3a–S3c online) and Fig. 4 lists the top ten hits.


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)

Analysis of negative control data. A, Multiple parallel affinity purifications of 25 nontagged lines (negative controls) and the use of ProteinCenter™ identified contaminants specific to FLAG and StrepII resins (FLAGome and STREPome respectively) and those common to both (BEADome). After identifying and removing the single protein hits (subsets) for each affinity method there is 72% overlap. B, The top ten hits for the FLAGome, STREPome and BEADome are listed in the table and the complete listings are presented in supplemental Tables S3A–S3C online. The top ten interacting proteins common to multiple baits are listed as the TRAPome and the complete list is presented in supplemental Table S5 online.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Analysis of negative control data. A, Multiple parallel affinity purifications of 25 nontagged lines (negative controls) and the use of ProteinCenter™ identified contaminants specific to FLAG and StrepII resins (FLAGome and STREPome respectively) and those common to both (BEADome). After identifying and removing the single protein hits (subsets) for each affinity method there is 72% overlap. B, The top ten hits for the FLAGome, STREPome and BEADome are listed in the table and the complete listings are presented in supplemental Tables S3A–S3C online. The top ten interacting proteins common to multiple baits are listed as the TRAPome and the complete list is presented in supplemental Table S5 online.
Mentions: MASCOT results were imported the into the ProteinCenter™ software package (see methods) to compare the FLAG and StrepII interaction data sets, compare Mascot protein probability scores of proteins identified in controls and test samples, and subtract nonspecific proteins that were pulled down in nontagged lines and no-protein systematic controls (supplemental Fig. S2). The software was also used to compare proteins from multiple pull-downs and highlight contaminants specific to each or both resins. Proteins consistently identified with FLAG resins only (FLAGome), StrepII resins only (STREPome) and those binding to both resins (BEADome) are listed in full (supplemental Tables S3a–S3c online) and Fig. 4 lists the top ten hits.

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