Glutathione is a small molecule – c-L-glutamyl-L-cysteinyl-glycine exists in either its reduced form, GSH, or the oxidized form, GSSG. Two glutathione molecules can be linked by disulfide bonds to form GSSG. Normally found in most cells as GSH, glutathione is also thought to play an important role in many processes within plant cells, including flowering, cell differentiation, programmed cell death, pathogen resistance, symbiosis, or cell cycle regulation.
Recent research has found that glutathione is involved in the post-translational modification process known as glutathionylation. S-thiolation is a disulfide compound formed between thiols and cysteine thiols, and the glutathione reaction is the major form of the S-thiolation reaction. This modification occurs mainly under conditions of oxidative stress but may also play an important role under normal conditions, especially for the regeneration of several thiol peroxidases.
Glutathionylation may be an important redox signaling mechanism that allows cells to sense signals from harmful stress conditions and trigger appropriate responses and has been shown to be involved in the regulation of several signaling pathways. Given the potential importance of this modification in stress response and adaptation, a number of methods have been developed to identify and analyze glutathionylated proteins.
Several of the most common primary methods for detecting glutathionylated proteins
Biotinylated Glutathione
Biotinylated glutathione can be readily synthesized in vitro as the reduced form (BioGSH), the oxidized form (BioGSSG), and an analog of reduced glutathione, glutathione ethyl ester (GEE), which also yields membrane-permeable biotinylated glutathione (BioGEE). The biotin group of glutathione protein can be detected sensitively and effectively by methods such as non-reducing non-reducing western blot and biotin antibodies. Biotinylated glutathione can also be identified using in vivo glutathionylation proteomics.
Anti-Glutathione Antibodies
Glutathionylated protein can be detected using commercially available glutathione antibodies or by the 1D or 2D gel electrophoresis, immunoprecipitation, and cellular immunolocalization. Commercially available glutathione antibodies can be used to analyze individual proteins but are unsuitable for large-scale detection.
Reduction of Glutathionylated Proteins by GRX
Mechanistically, the protein solution is alkylated with N-ethylmaleimide (NEM), thus blocking all free thiols. After the reduction of mononuclear cysteine GRX, the newly obtained thiolamines were derivatized by NEM-biotin to label glutathionylated proteins in the solution. Glutathionylated proteins were then purified by affinity chromatography and identified by mass spectrometry.
Workflow of GRX reduction of glutathionylated proteins
Our Protein Glutathione Sites Identification Service
Creative Proteomics offers an efficient and professional glutathione site identification service for analyzing a wide range of eukaryotic and prokaryotic samples. We will take care of all aspects of the project, including protein extraction, proteolytic cleavage, acylated peptide enrichment, peptide separation, mass spectrometry analysis, raw mass spectrometry data analysis, and bioinformatics analysis.
Sample requirements
- Fresh animal tissue: ≥600 mg
- Fresh plant tissue: ≥6 g
- Cell culture: ≥1×107 cells/tube x 3 tubes
- Fungi and bacteria: ≥600 mg
- Serum, plasma: 450 μL × 4 tubes
- Protein solution: total protein of 5-10 mg
- Body fluid samples: urine of 15 mL × 4 tubes (centrifuge at 1000 x g for 5 minutes and discard sediment); or other body fluids (saliva, amniotic fluid, cell culture supernatant, etc.) > 15 mL
Advantages
- High specificity and enrichment efficiency
- Large-scale identification of enriched glutathionylated peptides with mass spectrometry of high resolution and high throughput
- Combining commercially available quantitative techniques to analyze, compare, and correlate glutathionylation levels between samples quantitatively
Technology platforms
Ion Chromatography
High-Performance Liquid Chromatography (HPLC)
Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)