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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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Identification, coverage and reproducibility of the bait protein in parallel purifications. A, Peptide coverage for FLAG and StrepII purified baits (SGG) from two replicates and (B) reproducibility of the Mascot processed MS identification data for SGG. Peptide reproducibility % calculated from (No. peptides in common/No. total from 2 reps) × 100. M = oxidation of Met residue. C, Summary of the combined replicate bait data for the six different proteins analyzed. More detailed analysis is in supplemental Table S2 online and supplemental Fig. S7 online. MS identified the presence of three different isoforms for Rtnl1: PA and PD (both 25 kDa) and PF (64 kDa) and the % sequence coverage are shown respectively. Two different protein trap lines were analyzed for Fas2 and the data listed separately as (A) and (B).
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Figure 3: Identification, coverage and reproducibility of the bait protein in parallel purifications. A, Peptide coverage for FLAG and StrepII purified baits (SGG) from two replicates and (B) reproducibility of the Mascot processed MS identification data for SGG. Peptide reproducibility % calculated from (No. peptides in common/No. total from 2 reps) × 100. M = oxidation of Met residue. C, Summary of the combined replicate bait data for the six different proteins analyzed. More detailed analysis is in supplemental Table S2 online and supplemental Fig. S7 online. MS identified the presence of three different isoforms for Rtnl1: PA and PD (both 25 kDa) and PF (64 kDa) and the % sequence coverage are shown respectively. Two different protein trap lines were analyzed for Fas2 and the data listed separately as (A) and (B).

Mentions: To demonstrate the efficiency and reproducibility of our iPAC approach, six proteins with membrane, cytoplasmic or extracellular localizations, and molecular weights ranging from 22 to 2446 kDa, were studied in detail (Flapwing (FLW), Shaggy (SGG), Reticulon-like 1 (RTNL1), Fasciclin 2 (FAS2), Semaphorin 1a (SEMA-1A), and Dumpy (DP)): Fig. 3 and supplemental Table S2. Following affinity purification, MS analysis positively identified at least 2 peptides with all baits and up to 60% sequence coverage. In the examples shown, an average of 14 peptides were identified per protein from FLAG purifications and nine peptides from StrepII purifications, similar to the eight per protein identified by Brunner and colleagues (26) using a shotgun approach. Importantly, the coverage results in these cases suggest that there is little degradation or cleavage during the purification of even large proteins such as DP. Mapping the observed peptides allowed an investigation of protein cleavage or degradation as truncated proteins are unlikely to interact normally. For example, in the case of SGG, MS showed no peptides mapping to the C terminus (Fig. 3A). Alternatively these C-terminal peptides may have modifications that precluded their identification in MASCOT database searching. The additional data showing peptides liberated by the fused YFP tag confirms the presence of the bait and also serves as a standard across multiple samples, which can give an approximation of tagged protein abundance based on spectral counts (supplemental Table S2).


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)

Identification, coverage and reproducibility of the bait protein in parallel purifications. A, Peptide coverage for FLAG and StrepII purified baits (SGG) from two replicates and (B) reproducibility of the Mascot processed MS identification data for SGG. Peptide reproducibility % calculated from (No. peptides in common/No. total from 2 reps) × 100. M = oxidation of Met residue. C, Summary of the combined replicate bait data for the six different proteins analyzed. More detailed analysis is in supplemental Table S2 online and supplemental Fig. S7 online. MS identified the presence of three different isoforms for Rtnl1: PA and PD (both 25 kDa) and PF (64 kDa) and the % sequence coverage are shown respectively. Two different protein trap lines were analyzed for Fas2 and the data listed separately as (A) and (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Identification, coverage and reproducibility of the bait protein in parallel purifications. A, Peptide coverage for FLAG and StrepII purified baits (SGG) from two replicates and (B) reproducibility of the Mascot processed MS identification data for SGG. Peptide reproducibility % calculated from (No. peptides in common/No. total from 2 reps) × 100. M = oxidation of Met residue. C, Summary of the combined replicate bait data for the six different proteins analyzed. More detailed analysis is in supplemental Table S2 online and supplemental Fig. S7 online. MS identified the presence of three different isoforms for Rtnl1: PA and PD (both 25 kDa) and PF (64 kDa) and the % sequence coverage are shown respectively. Two different protein trap lines were analyzed for Fas2 and the data listed separately as (A) and (B).
Mentions: To demonstrate the efficiency and reproducibility of our iPAC approach, six proteins with membrane, cytoplasmic or extracellular localizations, and molecular weights ranging from 22 to 2446 kDa, were studied in detail (Flapwing (FLW), Shaggy (SGG), Reticulon-like 1 (RTNL1), Fasciclin 2 (FAS2), Semaphorin 1a (SEMA-1A), and Dumpy (DP)): Fig. 3 and supplemental Table S2. Following affinity purification, MS analysis positively identified at least 2 peptides with all baits and up to 60% sequence coverage. In the examples shown, an average of 14 peptides were identified per protein from FLAG purifications and nine peptides from StrepII purifications, similar to the eight per protein identified by Brunner and colleagues (26) using a shotgun approach. Importantly, the coverage results in these cases suggest that there is little degradation or cleavage during the purification of even large proteins such as DP. Mapping the observed peptides allowed an investigation of protein cleavage or degradation as truncated proteins are unlikely to interact normally. For example, in the case of SGG, MS showed no peptides mapping to the C terminus (Fig. 3A). Alternatively these C-terminal peptides may have modifications that precluded their identification in MASCOT database searching. The additional data showing peptides liberated by the fused YFP tag confirms the presence of the bait and also serves as a standard across multiple samples, which can give an approximation of tagged protein abundance based on spectral counts (supplemental Table S2).

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