In vitro determination of protein conjugates in human cells by LC-ESI-MS/MS after benzyl isothiocyanate exposure
Glucosinolates and their breakdown products, especially isothiocyanates (ITC), are hypothesized to exert a broad range of bioactivity. However, physiological mechanisms are not yet completely understood. In this study, formation of protein conjugates after incubation with benzyl isothiocyanate (BITC) was investigated in vitro. Survey of protein conjugates was done by determining BITC cysteine and lysine amino acid conjugates after protein digestion. Therefore, a liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated. Stability studies showed that cysteine conjugates are not stable under alkaline conditions, whereas lysine conjugates did not show any correlation to pH values, although stability increased at low temperatures. Lysine conjugates were the preferred form of protein conjugates and longer BITC exposure times led to higher amounts. Knowledge about the reaction sites of ITC in eukaryotic cells may help to understand the mode of action of ITC leading to health promoting as well as toxicological effects in humans.
Kuehn, C.; von Oesen, T.; Herz, C.; Schreiner, M.; Hanschen, F.S.; Lamy, E.; Rohn, S. 2018. In vitro determination of protein conjugates in human cells by LC-ESI-MS/MS after benzyl isothiocyanate exposure. Journal of Agricultural and Food Chemistry 66, 6727-6733.