Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.
Nat Protoc
; 8(6): 1155-68, 2013 Jun.
Article
em En
| MEDLINE
| ID: mdl-23702832
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Cromatografia Líquida
/
Complexo de Endopeptidases do Proteassoma
/
Espectrometria de Massas em Tandem
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Holanda