Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation
Fuzhong Ouyang , Tianrong Yu , Chao Gu , Guanghui Wang , Rui Shi , Rui Lv , Enhui Wu , Chongqing Ma , Ruochen Guo , Jing Li , Anna Żaczek , Jian Liu
AbstractThere is a great need to develop sensitive and speciﬁc methods for quantitative analysis of caspase-3 activities in cell apoptosis. Herein, we report a new method for sensitive detection of caspase-3 enzyme activities and inhibitor screening based on dual maleimide (DuMal) labeling quantitation and matrixassisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-TOF MS). Evaluation of caspase3 activities is performed using MS analysis of the enzymatic product of the peptide probe, which fuses a caspase-3 cleavable peptide segment (DEVD) and a quantiﬁable “ID tag” (a peptide segment of FRGLRGFKC labeled by maleimide). The DuMal labeling technique features non-isotopic tagging, rapid reactions, and reproducible quantitation. We have achieved quantitative analysis of the enzyme activities with a limit of detection (LOD) and limit of quantitation (LOQ) of caspase-3 down to 0.11 nM and 0.29 nM respectively and a proof-ofconcept demonstration of its inhibitor screening. Our method has further been tested for caspase-3 activities in a Parkinson’s disease cellular model, suggesting a useful tool for protease activity research.
|Journal series||Analyst, [The Analyst], ISSN 0003-2654, e-ISSN 1364-5528, (N/A 100 pkt)|
|Publication size in sheets||0.5|
|ASJC Classification||; ; ; ;|
|Score||= 100.0, 28-01-2020, ArticleFromJournal|
|Publication indicators||: 2018 = 0.945; : 2018 = 4.019 (2) - 2018=3.857 (5)|
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