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Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation

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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 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Evaluation of caspase-3 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-of-concept 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.

Graphical abstract: Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation

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Publication details

The article was received on 01 Aug 2019, accepted on 24 Sep 2019 and first published on 25 Sep 2019


Article type: Paper
DOI: 10.1039/C9AN01458F
Analyst, 2019, Advance Article

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    Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation

    F. Ouyang, T. Yu, C. Gu, G. Wang, R. Shi, R. Lv, E. Wu, C. Ma, R. Guo, J. Li, A. Zaczek and J. Liu, Analyst, 2019, Advance Article , DOI: 10.1039/C9AN01458F

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