UHPLC-HRMS-Based Analysis of S-Hydroxymethyl-Glutathione, GSH, and GSSG in Human Cells.
María Eugenia MongeManuela R MartinefskiMariela BolliniLucas B PontelPublished in: Methods in molecular biology (Clifton, N.J.) (2023)
Glutathione (GSH) is one of the main antioxidant molecules present in cells. It harbors a thiol group responsible for sustaining cellular redox homeostasis. This moiety can react with cellular electrophiles such as formaldehyde yielding the compound S-hydroxymethyl-GSH (HSMGSH). HSMGSH is the substrate of the enzyme alcohol dehydrogenase 5 (ADH5) and thus a key intermediate in formaldehyde metabolism. In this work, we describe a method for the chemical synthesis of HSMGSH and a pipeline to identify this compound in complex cell extracts by means of ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS). This method also allows determining GSH and oxidized disulfide (GSSG) in the same samples, thus providing broad information about formaldehyde-GSH metabolism.
Keyphrases
- high resolution mass spectrometry
- ultra high performance liquid chromatography
- tandem mass spectrometry
- fluorescent probe
- high resolution
- liquid chromatography
- gas chromatography
- ms ms
- simultaneous determination
- room temperature
- solid phase extraction
- mass spectrometry
- induced apoptosis
- single cell
- cell death
- cell therapy
- oxidative stress
- social media
- signaling pathway
- ionic liquid
- anti inflammatory
- high speed