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

Abstract

There 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.
Author Fuzhong Ouyang
Fuzhong Ouyang,,
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, Tianrong Yu
Tianrong Yu,,
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, Chao Gu
Chao Gu,,
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, Guanghui Wang
Guanghui Wang,,
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, Rui Shi
Rui Shi,,
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, Rui Lv
Rui Lv,,
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, Enhui Wu
Enhui Wu,,
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, Chongqing Ma
Chongqing Ma,,
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, Ruochen Guo
Ruochen Guo,,
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, Jing Li
Jing Li,,
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et al.`
Journal seriesAnalyst, [The Analyst], ISSN 0003-2654, e-ISSN 1364-5528, (N/A 100 pkt)
Issue year2019
Vol144
No22
Pages6751-6759
Publication size in sheets0.5
ASJC Classification1303 Biochemistry; 1602 Analytical Chemistry; 1603 Electrochemistry; 1607 Spectroscopy; 2304 Environmental Chemistry
DOIDOI:10.1039/C9AN01458F
URL https://pubs.rsc.org/en/content/articlepdf/2019/an/c9an01458f
Languageen angielski
Score (nominal)100
Score sourcejournalList
ScoreMinisterial score = 100.0, 28-01-2020, ArticleFromJournal
Publication indicators Scopus SNIP (Source Normalised Impact per Paper): 2018 = 0.945; WoS Impact Factor: 2018 = 4.019 (2) - 2018=3.857 (5)
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