Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells

Free Radic Biol Med. 2017 Nov;112:360-375. doi: 10.1016/j.freeradbiomed.2017.08.008. Epub 2017 Aug 12.


Glutathione (GSH) is the major non-protein thiol in humans and other mammals, which is present in millimolar concentrations within cells, but at much lower concentrations in the blood plasma. GSH and GSH-related enzymes act both to prevent oxidative damage and to detoxify electrophiles. Under oxidative stress, two GSH molecules become linked by a disulphide bridge to form glutathione disulphide (GSSG). Therefore, assessment of the GSH/GSSG ratio may provide an estimation of cellular redox metabolism. Current evidence resulting from studies in human blood, solid tissues, and cultured cells suggests that GSH also plays a prominent role in protein redox regulation via S -glutathionylation, i.e., the conjugation of GSH to reactive protein cysteine residues. A number of methodologies that enable quantitative analysis of GSH/GSSG ratio and S-glutathionylated proteins (PSSG), as well as identification and visualization of PSSG in tissue sections or cultured cells are currently available. Here, we have considered the main methodologies applied for GSH, GSSG and PSSG detection in biological samples. This review paper provides an up-to-date critical overview of the application of the most relevant analytical, morphological, and proteomics approaches to detect and analyse GSH, GSSG and PSSG in mammalian samples as well as discusses their current limitations.

Keywords: Anti-GSH antibodies; Biotinylation; Clickable glutathione; Fluorescent probes; Glutathione; Proteomics; S-glutathionylated proteins; Thiol alkylation.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Animals
  • Biotinylation
  • Blood Proteins / isolation & purification*
  • Blood Proteins / metabolism
  • Chromatography, High Pressure Liquid
  • Cysteine / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescent Dyes / chemistry
  • Glutathione / analysis
  • Glutathione / metabolism*
  • Glutathione Disulfide / analysis
  • Glutathione Disulfide / metabolism*
  • Humans
  • Liver / chemistry
  • Liver / metabolism
  • Oxidative Stress
  • Protein Processing, Post-Translational*
  • Proteomics / instrumentation
  • Proteomics / methods*
  • Tandem Mass Spectrometry


  • Blood Proteins
  • Fluorescent Dyes
  • Glutathione
  • Cysteine
  • Glutathione Disulfide