title: A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study.
abstract: We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity-based probes based on all seven isopeptide-linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage-specific manner.
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute (NKI), Plesmanlaan 121, 1066 CX Amsterdam (The Netherlands) http://research.nki.nl/Ovaalab/
authors.forename: Monique P C
authors.initials: MP
authors.lastname: Mulder
authors.forename: Farid
authors.initials: F
authors.lastname: El Oualid
authors.forename: Jarno
authors.initials: J
authors.lastname: ter Beek
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D004586:Electrophoresis; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D051379:Mice; D013379:Substrate Specificity; D014452:Ubiquitins / Q000737:chemistry*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; C109184:UBD protein, human; D014452:Ubiquitins; C085321:cyanine dye 5; D010450:Endopeptidases; C476458:OTUD7B protein, human
keywords: activity-based probes; native chemical ligation; protein modifications; solid-phase peptide synthesis; ubiquitin