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Proteomic Experiments #4

Detection of Diethylcarbamoyl-Cysteine following Acid Hydrolysis using Size Exclusion Liquid Chromatography-Tandem Mass Spectrometry

John C. L. Erve, Venkataraman Amarnath, Holly S. Valentine, Elizabeth G. Tonkin and William M. Valentine

Currently no analytical method is available to quantify the amount of diethylcarbamoyl-Cys formed on proteins following exposure to reactive metabolites of the drug disulfiram. We report here the development and validation of a semi-quantitiative LC/MS/MS method for the measurement of diethylcarbamoyl-Cys on protein following release by acid hydrolysis. After acid hydrolysis of protein,amino acid hydrolysates were spiked with the internal standard and separated by size exclusion chromatography. DETC-Cys was well separated from all other amino acids and were quantified using positive atmospheric pressure chemical ionization in a triple quandrupole mass spectrometer operating in the selected reaction monitoring mode.

The transitions m/z 221 to 100 arrising from loss of the diethylcarbamoyl moiety upon collision induced disociation was monitored. Calibration curves, which were linear over the range of 0 to 1000 ng diethylcarbamoyl-Cys on column, were analyzed along with each batch of samples. The assay was validated by running low, medium and high quality control samples. The intra- and inter-assay variability of diethylcarbamoyl-Cys ranged from -14 to 15%. The method was successfully applied to measure diethylcarbamoyl-Cys formed on rat hemoglobin following in vivo exposure to disulfiram.

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