title: A novel immuno-competitive capture mass spectrometry strategy for protein-protein interaction profiling reveals that LATS kinases regulate HCV replication through NS5A phosphorylation.
abstract: Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.
authors.affiliation: From the ‡Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Basel 4070, Switzerland and.
authors.forename: Hélène
authors.initials: H
authors.lastname: Meistermann
authors.affiliation: the ¶Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Nutley, NJ, 07110-1199.
authors.forename: Junjun
authors.initials: J
authors.lastname: Gao
authors.affiliation: From the ‡Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Basel 4070, Switzerland and.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Golling
authors.affiliation: From the ‡Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Basel 4070, Switzerland and.
authors.forename: Jens
authors.initials: J
authors.lastname: Lamerz
authors.affiliation: the ¶Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Nutley, NJ, 07110-1199.
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Pogam
authors.affiliation: From the ‡Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Basel 4070, Switzerland and.
authors.forename: Manuel
authors.initials: M
authors.lastname: Tzouros
authors.affiliation: the ¶Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Nutley, NJ, 07110-1199.
authors.forename: Sailaja
authors.initials: S
authors.lastname: Sankabathula
authors.affiliation: the ¶Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Nutley, NJ, 07110-1199.
authors.forename: Lore
authors.initials: L
authors.lastname: Gruenbaum
authors.affiliation: the ¶Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Nutley, NJ, 07110-1199.
authors.forename: Isabel
authors.initials: I
authors.lastname: Nájera
authors.affiliation: From the ‡Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Basel 4070, Switzerland and.
authors.forename: Hanno
authors.initials: H
authors.lastname: Langen
authors.affiliation: the ¶Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Nutley, NJ, 07110-1199.
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
authors.affiliation: From the ‡Pharma Research and Early Development Department, F. Hoffmann-La Roche Ltd, Basel 4070, Switzerland and angelique.augustin@roche.com.
authors.forename: Angélique
authors.initials: A
authors.lastname: Augustin
mesh_terms: D002460:Cell Line; D004261:DNA Replication / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000254:growth & development*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D015778:Minor Histocompatibility Antigens; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000032:analysis; D034622:RNA Interference; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase
chemical_list: D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D017361:Viral Nonstructural Proteins; C092745:LATS1 protein, human; C406449:LATS2 protein, human; C402182:phosphatidylinositol phosphate 4-kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase