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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

Effects of tags on protein levels. A, Protein extracts from heterozygous flies with insertions in Fer1LCH, Fer2HCH and Trailer hitch probed with Fer1LCH, Fer2HCH and Trailer hitch antibodies respectively to show the abundance of the tagged proteins (closed arrows) compared with the untagged proteins (open arrows). For a perfectly spliced and stable protein, the two band intensities would be expected to be equal. For Fer1HCH and Fer2LCH, tagged protein levels were 72 and 73% of the untagged, whereas for Trailer Hitch it was 19%. For Fer1HCH and Trailer hitch more than one fly line was available containing variants of the tag inserted; the figures are averaged from these lines. All these genes are predicted to encode single transcripts and protein products. Antibodies for Fer1LCH and Fer2HCH cross react with other proteins and extracts from untagged flies are run in adjacent lanes for comparison. B, Protein extracts from flies containing traps in growl and Rtnl1 (arrows indicate multiple isoforms) probed with anti-GFP to compare tagged protein levels with GFP, StrepII-tagged Venus YFP (SV), or FLAG-StrepII-tagged Venus YFP proteins. C, Confocal images comparing StrepII-tagged-venus proteins with (right) or without (left) FLAG. The addition of the FLAG tag does not reduce tagged protein levels. For these comparisons, the trap construct is inserted into the same intron and thus in the same position within the protein. Scale bars = 20 μm.
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Figure 2: Effects of tags on protein levels. A, Protein extracts from heterozygous flies with insertions in Fer1LCH, Fer2HCH and Trailer hitch probed with Fer1LCH, Fer2HCH and Trailer hitch antibodies respectively to show the abundance of the tagged proteins (closed arrows) compared with the untagged proteins (open arrows). For a perfectly spliced and stable protein, the two band intensities would be expected to be equal. For Fer1HCH and Fer2LCH, tagged protein levels were 72 and 73% of the untagged, whereas for Trailer Hitch it was 19%. For Fer1HCH and Trailer hitch more than one fly line was available containing variants of the tag inserted; the figures are averaged from these lines. All these genes are predicted to encode single transcripts and protein products. Antibodies for Fer1LCH and Fer2HCH cross react with other proteins and extracts from untagged flies are run in adjacent lanes for comparison. B, Protein extracts from flies containing traps in growl and Rtnl1 (arrows indicate multiple isoforms) probed with anti-GFP to compare tagged protein levels with GFP, StrepII-tagged Venus YFP (SV), or FLAG-StrepII-tagged Venus YFP proteins. C, Confocal images comparing StrepII-tagged-venus proteins with (right) or without (left) FLAG. The addition of the FLAG tag does not reduce tagged protein levels. For these comparisons, the trap construct is inserted into the same intron and thus in the same position within the protein. Scale bars = 20 μm.

Mentions: Author Contributions: JSR performed the proteomics experiments, analysed the data and drafted the manuscript. NL generated the constructs used and provided data for Figure 2 and supplemental Figure 4. IA assisted in the bioinformatics and prepared data sets for the public domain. ER performed sequencing and provided FlAnnotator. ED and HS generated, mapped and maintained the tagged D. melanogaster lines. KSL, SR and DStJ devised and supervised the project. Authors JSR, KSL, SR and DStJ wrote the manuscript and all authors approved it.


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)

Effects of tags on protein levels. A, Protein extracts from heterozygous flies with insertions in Fer1LCH, Fer2HCH and Trailer hitch probed with Fer1LCH, Fer2HCH and Trailer hitch antibodies respectively to show the abundance of the tagged proteins (closed arrows) compared with the untagged proteins (open arrows). For a perfectly spliced and stable protein, the two band intensities would be expected to be equal. For Fer1HCH and Fer2LCH, tagged protein levels were 72 and 73% of the untagged, whereas for Trailer Hitch it was 19%. For Fer1HCH and Trailer hitch more than one fly line was available containing variants of the tag inserted; the figures are averaged from these lines. All these genes are predicted to encode single transcripts and protein products. Antibodies for Fer1LCH and Fer2HCH cross react with other proteins and extracts from untagged flies are run in adjacent lanes for comparison. B, Protein extracts from flies containing traps in growl and Rtnl1 (arrows indicate multiple isoforms) probed with anti-GFP to compare tagged protein levels with GFP, StrepII-tagged Venus YFP (SV), or FLAG-StrepII-tagged Venus YFP proteins. C, Confocal images comparing StrepII-tagged-venus proteins with (right) or without (left) FLAG. The addition of the FLAG tag does not reduce tagged protein levels. For these comparisons, the trap construct is inserted into the same intron and thus in the same position within the protein. Scale bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effects of tags on protein levels. A, Protein extracts from heterozygous flies with insertions in Fer1LCH, Fer2HCH and Trailer hitch probed with Fer1LCH, Fer2HCH and Trailer hitch antibodies respectively to show the abundance of the tagged proteins (closed arrows) compared with the untagged proteins (open arrows). For a perfectly spliced and stable protein, the two band intensities would be expected to be equal. For Fer1HCH and Fer2LCH, tagged protein levels were 72 and 73% of the untagged, whereas for Trailer Hitch it was 19%. For Fer1HCH and Trailer hitch more than one fly line was available containing variants of the tag inserted; the figures are averaged from these lines. All these genes are predicted to encode single transcripts and protein products. Antibodies for Fer1LCH and Fer2HCH cross react with other proteins and extracts from untagged flies are run in adjacent lanes for comparison. B, Protein extracts from flies containing traps in growl and Rtnl1 (arrows indicate multiple isoforms) probed with anti-GFP to compare tagged protein levels with GFP, StrepII-tagged Venus YFP (SV), or FLAG-StrepII-tagged Venus YFP proteins. C, Confocal images comparing StrepII-tagged-venus proteins with (right) or without (left) FLAG. The addition of the FLAG tag does not reduce tagged protein levels. For these comparisons, the trap construct is inserted into the same intron and thus in the same position within the protein. Scale bars = 20 μm.
Mentions: Author Contributions: JSR performed the proteomics experiments, analysed the data and drafted the manuscript. NL generated the constructs used and provided data for Figure 2 and supplemental Figure 4. IA assisted in the bioinformatics and prepared data sets for the public domain. ER performed sequencing and provided FlAnnotator. ED and HS generated, mapped and maintained the tagged D. melanogaster lines. KSL, SR and DStJ devised and supervised the project. Authors JSR, KSL, SR and DStJ wrote the manuscript and all authors approved it.

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