Table 1.

Comparative methods to measure S-glutathionylation.

Direct methods			Indirect methods
Methods	Advantage	Disadvantage	Methods	Advantage	Disadvantage
	In vivo relevance.	Limited in multiplex capability to compare S-glutathionylation levels of an individual protein.		Most of the indirect methods have multiplex capability to compare S-glutathionylation levels of individual proteins.	Selective reduction strategy is not perfect in terms of specificity.
Anti-GSH antibodies	Easy to perform and compare global S-glutathionylation levels between different samples. When coupled with immunoprecipitation, the site of the modification can be identified.	Low sensitivities of these antibodies impart a limited detection potential and many of the proteins identified are abundant.	Radiolabel, fluorescent or biotin tag	Easy to perform and compare the global S-glutathionylation levels between different samples. When coupled with two dimensional gel and mass spectrometry, the site of the modification can be identified.	Does not offer the flexibility of quantification of S-glutathionylation levels of individual proteins in different samples.
[³⁵S] GSH	Radiolabeled S-glutathionylated proteins can be detected by radiography. When coupled with two dimensional gel and mass spectrometry, the site of the modification can be identified.	This method does not permit discrimination between proteins modified by [³⁵S]GSH and those ligated to [³⁵S]cysteines.. In addition, it requires preincubation with cycloheximide to block protein synthesis, likely perturbing cell physiology.	Isotopically labeled azido-biotin and ICAT	Allows for enrichment of S-glutathionylated proteins on avidin and offers the flexibility of quantification of S-glutathionylation levels of individual proteins in 2 samples.	Time consuming, labor intensive and expensive. Requires mass spectrometry for identification.
Biotinlated GSSG or bintinlated GSH ethyl esters	Allows for enrichment of S-glutathionylated proteins on streptavidin.	The bulky nature of biotin is likely to interfere with enzyme-mediated (de)glutathionylation and the artificial introduction of biotinylated GSH or GSSG may alter the endogenous redox status.	IodoTMT	Allows for enrichment of S-glutathionylated proteins with anti-TMT antibodies and offers the flexibility of multiplexed quantification of S-glutathionylation levels of individual proteins (6 samples).	Time consuming, labor intensive and expensive. Requires mass spectrometry for identification.
Clickable GSH	The small versatile azide handle seems not to interfere with enzyme-mediated (de) glutathionylation and can couple with a variety of different applications. No exogenous thiol is introduced.	The production of the azido-GSH is dependent on the transfection efficiencies of mutant glutathione synthetase.	Resin-assisted capture	The covalent capture by resin provides simpler workflow, higher enrichment specificity (>95%), and better sensitivity compared to the other indirect methods (ICAT and iodoTMT). It also offers the flexibility of multiplexed quantification of S-glutathionylation levels of individual proteins (4–10 samples).	Time consuming, labor intensive and expensive. Requires mass spectrometry for identification.