title: Label-free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL 36688, USA.
authors.forename: Dana V
authors.initials: DV
authors.lastname: Skarra
authors.forename: Marilyn
authors.initials: M
authors.lastname: Goudreault
authors.forename: Hyungwon
authors.initials: H
authors.lastname: Choi
authors.forename: Michael
authors.initials: M
authors.lastname: Mullin
authors.forename: Alexey I
authors.initials: AI
authors.lastname: Nesvizhskii
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.forename: Richard E
authors.initials: RE
authors.lastname: Honkanen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D018833:Chaperonins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D009687:Nuclear Proteins / Q000032:analysis* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D040901:Proteomics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; C079373:STIP1 protein, human; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; D018833:Chaperonins