title: Photoactivatable protein labeling by singlet oxygen mediated reactions.
abstract: Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158, USA; Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94158, USA.
authors.forename: Tsz-Leung
authors.initials: TL
authors.lastname: To
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158, USA.
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158, USA.
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158, USA; Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94158, USA.
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158, USA; Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94158, USA. Electronic address: Hyunil.jo@ucsf.edu.
authors.forename: Hyunil
authors.initials: H
authors.lastname: Jo
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158, USA; Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94158, USA. Electronic address: xiaokun.shu@ucsf.edu.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Shu
mesh_terms: D008958:Models, Molecular; D015394:Molecular Structure; D055668:Photochemical Processes; D017319:Photosensitizing Agents / Q000737:chemistry / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D026082:Singlet Oxygen / Q000378:metabolism*
chemical_list: D017319:Photosensitizing Agents; D044786:S-Phase Kinase-Associated Proteins; D026082:Singlet Oxygen
keywords: Biotinylation; Mass spectrometry; MiniSOG; Protein–protein interaction; Singlet oxygen