title: Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay.
abstract: Most mammalian tissues contain a single proteasome species: constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (β1c, β2c, β5c), the three homologous activities, β1i, β2i and β5i, may contain numerous distinct proteasome particles: immunoproteasomes (composed of β1i, β2i and β5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present.
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Gerjan
authors.initials: G
authors.lastname: de Bruin
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Bo-Tao
authors.initials: BT
authors.lastname: Xin
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D063347:Native Polyacrylamide Gel Electrophoresis; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D017433:Protein Structure, Secondary
chemical_list: D005456:Fluorescent Dyes; D061988:Proteasome Inhibitors; D046988:Proteasome Endopeptidase Complex