UHPLC-HRMS-Based Analysis of S-Hydroxymethyl-Glutathione, GSH, and GSSG in Human Cells

MONGE, MARÍA EUGENIA; MARTINEFSKI, MANUELA R.; BOLLINI, MARIELA; PONTEL, LUCAS B.

Methods in molecular biology (Clifton, N.J.)

NLM (Medline)

Lugar: Londres; Año: 2023 vol. 2675 p. 117 - 132

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.