Development of high-throughput quantitative analytical method for L-cysteine-containing dipeptides by LC-MS/MS toward its fermentative production.

L-Cysteine (Cys) is metabolically fundamental sulfur compound and important components in various cellular factors. Interestingly, free-form Cys itself as a simple monomeric amino acid was recently shown to function in a novel antioxidative system (cysteine/cystine shuttle system) in Escherichia coli. However, as for Cys-containing dipeptides, the biological functions, effects, and even contents have still remained largely elusive. The potential functions should be a part of cellular redox system and important in basic and applied biology. For its progress, establishment of reliable quantitation method is the first. However, such accurate analysis is unexpectedly difficult even in Cys, because thiol compounds convert through disulfide-exchange and air oxidation during sample preparation. Addressing this problem, in this study, thiol molecules like Cys-containing dipeptides were derivatized by using monobromobimane (thiol-specific alkylating reagent) and detected as S-bimanyl derivatives by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sample separation was processed with a C18 column (2.1 mm × 150 mm, 1.7 μm) and with water-acetonitrile gradient mobile phase containing 0.1% (v/v) formic acid at flow rate of 0.25 ml/min. The mass spectrometer was operated in the multiple reaction monitoring in positive/negative mode with electrospray ionization. The derivatization could indeed avoid the unfavorable reactions, namely, developed the method reflecting their correct contents on sampling. Furthermore, the method was successfully applied to monitoring Cys-containing dipeptides in E. coli Cys producer overexpressing bacD gene. This is the first report of the quantitative analysis of Cys-containing dipeptides, which should be useful for further study of fermentative production of Cys-containing dipeptides.