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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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GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.
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fig2: GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.

Mentions: An important issue for maximizing the identification of protein interaction partners is ensuring both efficient isolation of the target protein under study and achieving a high signal-to-noise ratio. In the case of FP-tagged proteins, our results show this is best achieved using the recently developed GFP binder (Rothbauer et al., 2008), which reproducibly provides near-quantitative depletion of GFP fusion proteins (Fig. 2). Direct comparison with commercially available anti-GFP monoclonal antibodies (mAbs) shows that an affinity matrix coupled to the GFP binder routinely produces higher depletion efficiencies and improves signal-to-noise ratios (Fig. 2, A and B; and unpublished data).


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)

GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.
© Copyright Policy
Related In: Results  -  Collection

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fig2: GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.
Mentions: An important issue for maximizing the identification of protein interaction partners is ensuring both efficient isolation of the target protein under study and achieving a high signal-to-noise ratio. In the case of FP-tagged proteins, our results show this is best achieved using the recently developed GFP binder (Rothbauer et al., 2008), which reproducibly provides near-quantitative depletion of GFP fusion proteins (Fig. 2). Direct comparison with commercially available anti-GFP monoclonal antibodies (mAbs) shows that an affinity matrix coupled to the GFP binder routinely produces higher depletion efficiencies and improves signal-to-noise ratios (Fig. 2, A and B; and unpublished data).

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
Related in: MedlinePlus