Issue 22, 2019

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

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

Supplementary files

Article information

Article type
Paper
Submitted
01 Aug 2019
Accepted
24 Sep 2019
First published
25 Sep 2019

Analyst, 2019,144, 6751-6759

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, 144, 6751 DOI: 10.1039/C9AN01458F

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