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, 11 (8), 1508-16

Label-free Quantitative Proteomics and SAINT Analysis Enable Interactome Mapping for the Human Ser/Thr Protein Phosphatase 5

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Label-free Quantitative Proteomics and SAINT Analysis Enable Interactome Mapping for the Human Ser/Thr Protein Phosphatase 5

Dana V Skarra et al. Proteomics.

Abstract

Affinity purification coupled to mass spectrometry (AP-MS) represents a powerful and proven approach for the analysis of protein-protein interactions. However, the detection of true interactions for proteins that are commonly considered background contaminants is currently a limitation of AP-MS. Here using spectral counts and the new statistical tool, Significance Analysis of INTeractome (SAINT), true interaction between the serine/threonine protein phosphatase 5 (PP5) and a chaperonin, heat shock protein 90 (Hsp90), is discerned. Furthermore, we report and validate a new interaction between PP5 and an Hsp90 adaptor protein, stress-induced phosphoprotein 1 (STIP1; HOP). Mutation of PP5, replacing key basic amino acids (K97A and R101A) in the tetratricopeptide repeat (TPR) region known to be necessary for the interactions with Hsp90, abolished both the known interaction of PP5 with cell division cycle 37 homolog and the novel interaction of PP5 with stress-induced phosphoprotein 1. Taken together, the results presented demonstrate the usefulness of label-free quantitative proteomics and statistical tools to discriminate between noise and true interactions, even for proteins normally considered as background contaminants.

Conflict of interest statement

Conflict of interest. None of the authors have a financial or commercial conflict of interest.

Figures

Figure 1
Figure 1. Schematics of the experimental and analytical pipeline
A) wt-PP5-FLAG, ΔTPR-PP5-FLAG or FLAG alone cells lines were generated, and subjected to AP-MS analysis in four biological replicates. The grey circle represents a non-specific interaction partner which associates to the FLAG alone, the orange circle represents an interaction partner for the wt and mutant PP5 which is not recovered in the FLAG alone purifications. The blue circle represents a protein that strongly interacts with wt but not ΔTPR PP5, yet can also be detected in lower abundance in purifications of the FLAG alone. B) Schematic representation of the spectral counts for the blue, orange and grey proteins across the four biological replicates. C) Typical results obtained using simple binary contaminant filtering, such as frequency filters or removal of all hits identified in the FLAG alone sample. While the orange and grey proteins are successfully identified as specific and background, respectively, the blue protein is erroneously labeled as a contaminant, resulting in a false-negative identification. D) SAINT utilizes a semi-supervised mixture model of the spectral count distribution of each protein across the negative control runs (blue line) and provides probability values that each bait-prey interaction is real.
Figure 2
Figure 2. Cytoscape representation of the wt-PP5 and Hsp90 interaction partners
AP-MS data is from Table I; all interaction partners have an AvgP≥0.5. The thickness of the lines is proportional to the total spectral counts for the hits in the four biological replicates purifications of the bait.
Figure 3
Figure 3. Hsp90 and Cdc37 interacts with wt-PP5, but not a TPR mutant or with the phosphatase PP4
Immunoprecipitation on anti-FLAG (M2 agarose) beads was performed on lysate from HEK293T cells transiently co-expressing the indicated FLAG- and GFP-tagged constructs. Immune complexes were resolved by SDS-PAGE followed by transfer to nitrocellulose. Co-precipitation of GFP-tagged proteins was detected by immunoblotting (IB) for the GFP tag (top panels; the positions of the tagged proteins are indicated by arrows). The precipitated FLAG-tagged protein was detected with anti-FLAG antibodies (bottom panels). Total protein lysate (left) was analyzed in parallel to the immunoprecipitation (right) to monitor protein expression.
Figure 4
Figure 4. STIP1 interacts with wt-PP5, but not a TPR mutant
Immunoprecipitation on anti-FLAG (M2 agarose) beads was performed on lysate from HEK293T cells transiently co-expressing the indicated FLAG- and GFP-tagged constructs. Immune complexes were resolved by SDS-PAGE followed by transfer to nitrocellulose. Co-precipitation of GFP-tagged proteins was detected by immunoblotting (IB) for the GFP tag (top panels; position of the tagged proteins are indicated by arrows). The precipitated FLAG-tagged protein was detected with anti-FLAG antibodies (bottom panels). Total protein lysate (left) was analyzed in parallel to the immunoprecipitation (right) to monitor protein expression.

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