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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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Comparison of bead proteomes. (A) Design of the SILAC immunoprecipitation experiment used to compare the bead proteomes of agarose, Sepharose, and magnetic beads. For all three, the protein G–conjugated versions were used. The experiment was performed in two stages, first with a short incubation time of 30 min and next with a long incubation time of 18 h. In addition, cells were fractionated into cytoplasmic and nuclear extracts to compare the profiles of the proteins that bind nonspecifically to the bead matrices. In the case of nuclear extracts, more proteins bind nonspecifically during a long incubation than a short incubation, as assessed both by Coomassie staining (B) and by mass spectrometric analysis (C). The cytoplasmic protein profile did not vary to the same extent. The distribution of proteins by class was quite similar regardless of the cellular extract used in the experiment or the time of incubation (C). Distinct differences in the distribution of these classes of proteins were observed, however, with magnetic beads binding more cytoskeletal and structural proteins nonspecifically and Sepharose binding more nucleic acid binding factors nonspecifically.
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fig3: Comparison of bead proteomes. (A) Design of the SILAC immunoprecipitation experiment used to compare the bead proteomes of agarose, Sepharose, and magnetic beads. For all three, the protein G–conjugated versions were used. The experiment was performed in two stages, first with a short incubation time of 30 min and next with a long incubation time of 18 h. In addition, cells were fractionated into cytoplasmic and nuclear extracts to compare the profiles of the proteins that bind nonspecifically to the bead matrices. In the case of nuclear extracts, more proteins bind nonspecifically during a long incubation than a short incubation, as assessed both by Coomassie staining (B) and by mass spectrometric analysis (C). The cytoplasmic protein profile did not vary to the same extent. The distribution of proteins by class was quite similar regardless of the cellular extract used in the experiment or the time of incubation (C). Distinct differences in the distribution of these classes of proteins were observed, however, with magnetic beads binding more cytoskeletal and structural proteins nonspecifically and Sepharose binding more nucleic acid binding factors nonspecifically.

Mentions: Using the SILAC protocol, a comparison was made of nonspecific protein binding to Sepharose as compared with two other commonly used affinity matrices, i.e., agarose and magnetic beads (Fig. 3). In this case, labeling was conducted using three isotopic states, i.e., 12C-arg and 12C-lys for agarose, 13C-arg and D4-lys for Sepharose, and 13C/15N-arg and 13C/15N-lys for magnetic beads. Nonspecific protein binding was observed for all three matrices after incubation of either nuclear or cytoplasmic extracts, whether the incubation time was short (30 min) or long (18 h). At both the short and long time points, a similar distribution of classes of contaminating proteins was observed, although the levels of protein binding can increase after longer incubation. An interesting difference was apparent in the relative performance of Sepharose and magnetic beads when incubated with either nuclear or cytoplasmic extracts. Thus, magnetic beads, which showed more nonspecific binding to structural/motility protein classes and lower nonspecific binding to nucleic acid–binding factors, had lower backgrounds of contaminating proteins in nuclear extracts as compared with Sepharose. In contrast, Sepharose, which showed more nonspecific interactions with nucleic acid–binding factors, gave better results than magnetic beads in reducing nonspecific background in cytoplasmic extracts (Fig. 3 C; Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200805092/DC1). In the case of agarose beads, similar levels of nonspecific binding to Sepharose were observed in nuclear extracts, whereas agarose beads showed lower nonspecific binding in cytoplasmic extracts as compared with either Sepharose or magnetic beads (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200805092/DC1). Overall, it can be concluded that the affinity matrices constitute a major source of nonspecific protein binding for all protein interaction studies and the detailed data obtained from comparing the three main types of affinity matrices show that no single type of bead is ideally suited to all applications. Rather, improved results with respect to nonspecific protein binding can be obtained by using different types of affinity matrix depending upon whether protein interaction studies are performed using cytoplasmic or nuclear extracts, or other types of cellular fractions.


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)

Comparison of bead proteomes. (A) Design of the SILAC immunoprecipitation experiment used to compare the bead proteomes of agarose, Sepharose, and magnetic beads. For all three, the protein G–conjugated versions were used. The experiment was performed in two stages, first with a short incubation time of 30 min and next with a long incubation time of 18 h. In addition, cells were fractionated into cytoplasmic and nuclear extracts to compare the profiles of the proteins that bind nonspecifically to the bead matrices. In the case of nuclear extracts, more proteins bind nonspecifically during a long incubation than a short incubation, as assessed both by Coomassie staining (B) and by mass spectrometric analysis (C). The cytoplasmic protein profile did not vary to the same extent. The distribution of proteins by class was quite similar regardless of the cellular extract used in the experiment or the time of incubation (C). Distinct differences in the distribution of these classes of proteins were observed, however, with magnetic beads binding more cytoskeletal and structural proteins nonspecifically and Sepharose binding more nucleic acid binding factors nonspecifically.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Comparison of bead proteomes. (A) Design of the SILAC immunoprecipitation experiment used to compare the bead proteomes of agarose, Sepharose, and magnetic beads. For all three, the protein G–conjugated versions were used. The experiment was performed in two stages, first with a short incubation time of 30 min and next with a long incubation time of 18 h. In addition, cells were fractionated into cytoplasmic and nuclear extracts to compare the profiles of the proteins that bind nonspecifically to the bead matrices. In the case of nuclear extracts, more proteins bind nonspecifically during a long incubation than a short incubation, as assessed both by Coomassie staining (B) and by mass spectrometric analysis (C). The cytoplasmic protein profile did not vary to the same extent. The distribution of proteins by class was quite similar regardless of the cellular extract used in the experiment or the time of incubation (C). Distinct differences in the distribution of these classes of proteins were observed, however, with magnetic beads binding more cytoskeletal and structural proteins nonspecifically and Sepharose binding more nucleic acid binding factors nonspecifically.
Mentions: Using the SILAC protocol, a comparison was made of nonspecific protein binding to Sepharose as compared with two other commonly used affinity matrices, i.e., agarose and magnetic beads (Fig. 3). In this case, labeling was conducted using three isotopic states, i.e., 12C-arg and 12C-lys for agarose, 13C-arg and D4-lys for Sepharose, and 13C/15N-arg and 13C/15N-lys for magnetic beads. Nonspecific protein binding was observed for all three matrices after incubation of either nuclear or cytoplasmic extracts, whether the incubation time was short (30 min) or long (18 h). At both the short and long time points, a similar distribution of classes of contaminating proteins was observed, although the levels of protein binding can increase after longer incubation. An interesting difference was apparent in the relative performance of Sepharose and magnetic beads when incubated with either nuclear or cytoplasmic extracts. Thus, magnetic beads, which showed more nonspecific binding to structural/motility protein classes and lower nonspecific binding to nucleic acid–binding factors, had lower backgrounds of contaminating proteins in nuclear extracts as compared with Sepharose. In contrast, Sepharose, which showed more nonspecific interactions with nucleic acid–binding factors, gave better results than magnetic beads in reducing nonspecific background in cytoplasmic extracts (Fig. 3 C; Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200805092/DC1). In the case of agarose beads, similar levels of nonspecific binding to Sepharose were observed in nuclear extracts, whereas agarose beads showed lower nonspecific binding in cytoplasmic extracts as compared with either Sepharose or magnetic beads (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200805092/DC1). Overall, it can be concluded that the affinity matrices constitute a major source of nonspecific protein binding for all protein interaction studies and the detailed data obtained from comparing the three main types of affinity matrices show that no single type of bead is ideally suited to all applications. Rather, improved results with respect to nonspecific protein binding can be obtained by using different types of affinity matrix depending upon whether protein interaction studies are performed using cytoplasmic or nuclear extracts, or other types of cellular fractions.

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