Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

TY - JOUR

T1 - Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

AU - Li, Nan

AU - Kuo, Chi-Lin

AU - Paniagua, Guillem

AU - Van Den Elst, Hans

AU - Verdoes, Martijn

AU - Willems, Lianne I.

AU - Van Der Linden, Wouter A.

AU - Ruben, Mark

AU - Van Genderen, Eric

AU - Gubbens, Jacob

AU - Van Wezel, Gilles P.

AU - Overkleeft, Herman S.

AU - Florea, Bogdan I.

PY - 2013/6

Y1 - 2013/6

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84879378806&partnerID=8YFLogxK

U2 - 10.1038/nprot.2013.065

DO - 10.1038/nprot.2013.065

M3 - Article

C2 - 23702832

AN - SCOPUS:84879378806

VL - 8

SP - 1155

EP - 1168

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

IS - 6

ER -