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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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Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.
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fig1: Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.

Mentions: A standard workflow for SILAC-based analysis of protein interaction partners in pull-down experiments is summarized in Fig. 1. In brief, the total protein components isolated from either an immunoprecipitation or affinity pull-down experiment are size fractionated using SDS-PAGE. The gel is cut into typically 5–10 slices, each of which is digested with trypsin and the resulting peptides eluted and analyzed by high sensitivity mass spectrometry (see Materials and methods).


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)

Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.
Mentions: A standard workflow for SILAC-based analysis of protein interaction partners in pull-down experiments is summarized in Fig. 1. In brief, the total protein components isolated from either an immunoprecipitation or affinity pull-down experiment are size fractionated using SDS-PAGE. The gel is cut into typically 5–10 slices, each of which is digested with trypsin and the resulting peptides eluted and analyzed by high sensitivity mass spectrometry (see Materials and methods).

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