biostudies-literatureTerasawa KResearch Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research [(BINDS])MEXT programGrants-in-Aid for Scientific ResearchGrants-in-Aids for ScientificJapan Agency for Medical Research and DevelopmentEstablishment of University Fellowships towards the Creation of Science Technology Innovatione4823https://www.ebi.ac.uk/biostudies/studies/S-EPMC10659947biostudies-literatureUnknown32(12)Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (N^ε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminProtein science : a publication of the Protein SocietySite-specific photo-crosslinking/cleavage for protein-protein interface identification reveals oligomeric assembly of lysosomal-associated membrane protein type 2A in mammalian cells.PMC10659947JP17K12005JP21K10063JPMJFS2109JP23ama121027Iwata TYokoyama STerasawa KSakamoto KTerada TWatabe TOhtake KSeike THara-Yokoyama MfalseSite-specific photo-crosslinking/cleavage for protein-protein interface identification reveals oligomeric assembly of lysosomal-associated membrane protein type 2A in mammalian cells.Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (N^ε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-termin2023-01-01T00:00:00Z2023 Dec2026-06-27T03:13:22.483Z2025-04-04T09:39:32.329ZS-EPMC106599473790669410.1002/pro.4823