Mass spectrometry based detection of glutathione with sensitivity for single-cell analysis

Rationale: Glutathione (GSH) is a very important molecule that participates in various physiologically important events. Depletion of cellular GSH has been suggested as a biomarker of early stage of cell injury. Assessment of GSH with high selectivity and sensitivity is in demand in many fields of life sciences.

Methods: Cell lysates were deproteinated by 5-sulfosalicylic acid. Glutathione was derivatized with monobromobimane to form a GSH conjugate. S-Hexylglutathione was employed as internal standard. The resulting samples were analyzed using a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) system.

Results: The limit of detection was as low as 500 amol with high selectivity. The approach was sensitive enough to detect GSH content in a single human erythrocyte. The technique is the most sensitive approach among the reported MS-based GSH assays.

Conclusions: A monobromobimane derivatization and mass spectrometry based method was developed for the assessment of GSH. The derivatized GSH was chemically stable against autooxidation. With the great selectivity and sensitivity, the approach is anticipated to serve as a routine method for assessment of GSH and possibly other important endogenous low molecular weight thiols.