title: A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.
abstract: 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.
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Kaake
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.affiliation: ¶Department of Chemistry, University of California, Irvine, California 92697;
authors.forename: Anthony
authors.initials: A
authors.lastname: Burke
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
authors.forename: Clinton
authors.initials: C
authors.lastname: Yu
authors.affiliation: ¶Department of Chemistry, University of California, Irvine, California 92697;
authors.forename: Wynne
authors.initials: W
authors.lastname: Kandur
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
authors.forename: Yingying
authors.initials: Y
authors.lastname: Yang
authors.affiliation: ¶Department of Chemistry, University of California, Irvine, California 92697;
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Novtisky
authors.affiliation: ‖Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134;
authors.forename: Tonya
authors.initials: T
authors.lastname: Second
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
authors.forename: Jicheng
authors.initials: J
authors.lastname: Duan
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
authors.forename: Athit
authors.initials: A
authors.lastname: Kao
authors.affiliation: **Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143.
authors.forename: Shenheng
authors.initials: S
authors.lastname: Guan
authors.affiliation: ¶Department of Chemistry, University of California, Irvine, California 92697;
authors.forename: Danielle
authors.initials: D
authors.lastname: Vellucci
authors.affiliation: ¶Department of Chemistry, University of California, Irvine, California 92697;
authors.forename: Scott D
authors.initials: SD
authors.lastname: Rychnovsky
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697; lanhuang@uci.edu.
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D045304:Cytochromes c / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D020543:Proteome / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D020543:Proteome; D001710:Biotin; D045304:Cytochromes c