A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells
2014; Elsevier BV; Volume: 13; Issue: 12 Linguagem: Inglês
10.1074/mcp.m114.042630
ISSN1535-9484
AutoresRobyn M. Kaake, Xiaorong Wang, Anthony M. Burke, Clinton Yu, Wynne V. Kandur, Yingying Yang, Eric J. Novtisky, Tonya Second, Jicheng Duan, Athit Kao, Shenheng Guan, Danielle Vellucci, Scott D. Rychnovsky, Lan Huang,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoProtein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems. Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems. Protein–protein interactions (PPIs) 1The abbreviations used are:PPIprotein–protein interactionXL-MScross-linking mass spectrometryAP-MSaffinity purification mass spectrometryAzide-A-DSBSOazide-tagged, acid-cleavable disuccinimidyl bis-sulfoxideBARACbiarylazacycloctynoneHBHis-BioDSSOdisuccinimidyl sulfoxideMSmass spectrometryMS2tandem mass spectrometryMSnmulti-stage tandem mass spectrometryLCliquid chromatographyTPCKL-1-tosylamido-2-phenylethyl chloromethyl ketone.1The abbreviations used are:PPIprotein–protein interactionXL-MScross-linking mass spectrometryAP-MSaffinity purification mass spectrometryAzide-A-DSBSOazide-tagged, acid-cleavable disuccinimidyl bis-sulfoxideBARACbiarylazacycloctynoneHBHis-BioDSSOdisuccinimidyl sulfoxideMSmass spectrometryMS2tandem mass spectrometryMSnmulti-stage tandem mass spectrometryLCliquid chromatographyTPCKL-1-tosylamido-2-phenylethyl chloromethyl ketone. play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1Ryan D.P. Matthews J.M. Protein-protein interactions in human disease.Curr. Opin. Struct. Biol. 2005; 15: 441-446Crossref PubMed Scopus (245) Google Scholar). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics. protein–protein interaction cross-linking mass spectrometry affinity purification mass spectrometry azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide biarylazacycloctynone His-Bio disuccinimidyl sulfoxide mass spectrometry tandem mass spectrometry multi-stage tandem mass spectrometry liquid chromatography L-1-tosylamido-2-phenylethyl chloromethyl ketone. protein–protein interaction cross-linking mass spectrometry affinity purification mass spectrometry azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide biarylazacycloctynone His-Bio disuccinimidyl sulfoxide mass spectrometry tandem mass spectrometry multi-stage tandem mass spectrometry liquid chromatography L-1-tosylamido-2-phenylethyl chloromethyl ketone. Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2Gingras A.C. Gstaiger M. Raught B. Aebersold R. Analysis of protein complexes using mass spectrometry.Nat. Rev. Mol. Cell Biol. 2007; 8: 645-654Crossref PubMed Scopus (558) Google Scholar, 3Kocher T. Superti-Furga G. Mass spectrometry-based functional proteomics: from molecular machines to protein networks.Nat. Methods. 2007; 4: 807-815Crossref PubMed Scopus (186) Google Scholar, 4Guan H. Kiss-Toth E. Advanced technologies for studies on protein interactomes.Adv. Biochem. Eng. Biotechnol. 2008; 110: 1-24PubMed Google Scholar, 5Ryan C.J. Cimermancic P. Szpiech Z.A. Sali A. Hernandez R.D. Krogan N.J. High-resolution network biology: connecting sequence with function.Nat. Rev. Genet. 2013; 14: 865-879Crossref PubMed Scopus (66) Google Scholar). 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To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10Vasilescu J. Guo X. Kast J. Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.Proteomics. 2004; 4: 3845-3854Crossref PubMed Scopus (190) Google Scholar, 11Schmitt-Ulms G. Hansen K. Liu J. Cowdrey C. Yang J. DeArmond S.J. Cohen F.E. Prusiner S.B. Baldwin M.A. Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.Nat. Biotechnol. 2004; 22: 724-731Crossref PubMed Scopus (136) Google Scholar, 12Guerrero C. Tagwerker C. Kaiser P. Huang L. An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network.Mol. Cell. 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Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSn). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level. Bovine cytochrome C (>95% purity) was purchased from Sigma Aldrich (St. Louis, MO). Amicon Ultra 100-kDa, 30-kDa, and 10-kDa NMWL centrifugal filters were purchased from EMD Millipore (Billerica, MA). LaminA/C antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA). Calnexin and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Streptavidin agarose resin, high-capacity streptavidin agarose resin, HRP-conjugated streptavidin, and Super Signal West Pico chemiluminescent substrate were purchased from Thermo Scientific (Rockford, IL). Sequencing-grade trypsin was purchased from Promega Corp. (Madison, WI). Endoproteinase Lys-C was purchased from WAKO Chemicals (Osaka, Japan). TPCK-treated trypsin was purchased from Worthington Biochemical Corp (Lakewood, NJ). All other general chemicals for buffers and culture media were purchased from Fisher Scientific or VWR International (Radnor, PA). The synthesis and characterization of the Azide-A-DSBSO cross-linker are described in Ref. 55Burke A.M. Ph.D. thesis. Reagents for in vivo Protein Cross-Linking and Automated Analysis of Protein-Protein Interactions with Tandem Mass Spectrometry University of California, Irvine2011Google Scholar. The simplified scheme is depicted in Fig. 1. BARAC reagent was synthesized as described elsewhere (45Jewett J.C. Sletten E.M. Bertozzi C.R. Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones.J. Am. Chem. Soc. 2010; 132: 3688-3690Crossref PubMed Scopus (523) Google Scholar). Protein samples were separated via SDS-PAGE and either stained using Coomassie Blue or transferred to a PVDF membrane and analyzed via immunoblotting. Biotin-conjugated proteins and HB-tagged proteins were detected by streptavidin-HRP conjugate. Cross-linked and non-cross-linked Rpn11-HB and HB-Rpt6 were also detected with streptavidin-HRP conjugate. Lamin A/C, calnexin, and GAPDH were detected using specific primary antibodies and either rabbit or mouse secondary HRP-conjugated antibody. Biotin-conjugated peptides were blotted onto nitrocellulose membrane and detected with streptavidin-HRP conjugate. Azide-A-DSBSO cross-linking of bovine cytochrome C was similar to that described elsewhere (35Vellucci D. Kao A. Kaake R.M. Rychnovsky S.D. Huang L. Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry.J. Am. Soc. Mass Spectrom. 2010; 21: 1432-1445Crossref PubMed Scopus (33) Google Scholar). The reaction was quenched with 500 mm NH4HCO3, and samples were ultracentrifuged on 10-kDa NMWL Amicon Ultra centrifugal filters to remove excess cross-linker. Various amounts of BARAC were then reacted with the cross-linked cytochrome C in either phosphate or 8 m urea lysis buffer with agitation overnight. The reaction efficiency for each condition was evaluate
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