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Methods Mol Biol. 2019;1990:27-42. doi: 10.1007/978-1-4939-9463-2_3.

Investigating ROS, RNS, and H2S-Sensitive Signaling Proteins.

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Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK.
Horizon Discovery Ltd., Cambridge, UK.
Medical School Building, University of Exeter, Exeter, UK.
Geoffrey Pope Building, University of Exeter, Exeter, UK.
Faculty of Agrobiotechnical Sciences, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia.
Department of Applied Sciences, University of the West of England, Bristol, UK.


The modification of proteins is a key way to alter their activity and function. Often thiols, cysteine residues, on proteins are attractive targets for such modification. Assuming that the thiol group is accessible then reactions may take place with a range of chemicals found in cells. These may include reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reactive nitrogen species such as nitric oxide (NO), hydrogen sulfide (H2S), or glutathione. Such modifications often are instrumental to important cellular signaling processes, which ultimately result in modification of physiology of the organism. Therefore, there is a need to be able to identify such modifications. There are a variety of techniques to find proteins which may be altered in this way but here the focus is on two approaches: firstly, the use of fluorescent thiol derivatives and the subsequent use of mass spectrometry to identify the thiols involved; secondly the confirmation of such changes using biochemical assays and genetic mutants. The discussion will be based on the use of two model organisms: firstly the plant Arabidopsis thaliana (both as cell cultures and whole plants) and secondly the nematode worm Caenorhabditis elegans. However, these tools, as described, may be used in a much wider range of biological systems, including human and human tissue cultures.


5′-Iodoacetamide fluorescein; Glutathione; Glyceraldehyde 3-phosphate dehydrogenase; Histidine kinase; Hydrogen peroxide; Hydrogen sulfide; Nitric oxide; Reactive nitrogen species; Reactive oxygen species; Stomatal guard cells; Thiol labeling

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