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Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.

Trinkle-Mulcahy L, Boulon S, Lam YW, Urcia R, Boisvert FM, Vandermoere F, Morrice NA, Swift S, Rothbauer U, Leonhardt H, Lamond A - J. Cell Biol. (2008)

Bottom Line: GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy.Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design.These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland, UK. ltrinkle@uottawa.ca

ABSTRACT
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

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Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).
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fig4: Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).

Mentions: Because SMN is found in multiprotein complexes in both the nucleus and the cytoplasm (Fig. 4 A), and because some of its previously identified interactions were reported to be compartment specific (Fig. 4 B), we fractionated cells into nuclear and cytoplasmic extracts to compare the interaction partners identified by SILAC in both compartments. A HeLa cell line stably expressing GFP-SMN (Sleeman et al., 2003) was grown in media containing 13C-labeled arginine and lysine, with parental HeLa cells grown in normal 12C-labeled media as a negative control. The cells were harvested and fractionated into cytoplasmic and nuclear extracts, pull-down experiments were performed using the GFP binder, and proteins were analyzed by mass spectrometry. This resulted in identification of over 20 proteins previously described to copurify with SMN. The average SILAC ratio and number of peptides identified for each protein in both cytoplasmic and nuclear extracts is listed in Table III.


Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.

Trinkle-Mulcahy L, Boulon S, Lam YW, Urcia R, Boisvert FM, Vandermoere F, Morrice NA, Swift S, Rothbauer U, Leonhardt H, Lamond A - J. Cell Biol. (2008)

Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568020&req=5

fig4: Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).
Mentions: Because SMN is found in multiprotein complexes in both the nucleus and the cytoplasm (Fig. 4 A), and because some of its previously identified interactions were reported to be compartment specific (Fig. 4 B), we fractionated cells into nuclear and cytoplasmic extracts to compare the interaction partners identified by SILAC in both compartments. A HeLa cell line stably expressing GFP-SMN (Sleeman et al., 2003) was grown in media containing 13C-labeled arginine and lysine, with parental HeLa cells grown in normal 12C-labeled media as a negative control. The cells were harvested and fractionated into cytoplasmic and nuclear extracts, pull-down experiments were performed using the GFP binder, and proteins were analyzed by mass spectrometry. This resulted in identification of over 20 proteins previously described to copurify with SMN. The average SILAC ratio and number of peptides identified for each protein in both cytoplasmic and nuclear extracts is listed in Table III.

Bottom Line: GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy.Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design.These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland, UK. ltrinkle@uottawa.ca

ABSTRACT
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

Show MeSH