title: Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.
abstract: Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre, Leiden, The Netherlands.
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.forename: Chi-Lin
authors.initials: CL
authors.lastname: Kuo
authors.forename: Guillem
authors.initials: G
authors.lastname: Paniagua
authors.forename: Hans
authors.initials: H
authors.lastname: van den Elst
authors.forename: Martijn
authors.initials: M
authors.lastname: Verdoes
authors.forename: Lianne I
authors.initials: LI
authors.lastname: Willems
authors.forename: Wouter A
authors.initials: WA
authors.lastname: van der Linden
authors.forename: Mark
authors.initials: M
authors.lastname: Ruben
authors.forename: Eric
authors.initials: E
authors.lastname: van Genderen
authors.forename: Jacob
authors.initials: J
authors.lastname: Gubbens
authors.forename: Gilles P
authors.initials: GP
authors.lastname: van Wezel
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D005453:Fluorescence; D015336:Molecular Probe Techniques; D015394:Molecular Structure; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011506:Proteins / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011506:Proteins; D046988:Proteasome Endopeptidase Complex