title: Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label.
abstract: Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein-protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey-bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein-protein interactions and enables simultaneous mapping of the binding interface under living conditions.
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Haiping
authors.initials: H
authors.lastname: Song
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Dan
authors.initials: D
authors.lastname: He
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Shizhong
authors.initials: S
authors.lastname: Dai
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Shixian
authors.initials: S
authors.lastname: Lin
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Meng
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.|Peking-Tsinghua Center for Life Sciences, Beijing 100871, China.
authors.forename: Chu
authors.initials: C
authors.lastname: Wang
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.|Peking-Tsinghua Center for Life Sciences, Beijing 100871, China.
authors.forename: Peng R
authors.initials: PR
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D013058:Mass Spectrometry; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D013194:Staining and Labeling
chemical_list: D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D011506:Proteins