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Assessing covalent binding of reactive drug metabolites by complete protein digestion and LC-MS analysis
Nicoleta Mitrea1, André LeBlanc, Maude St-Onge
1Laboratory for Bioanalytical Mass Spectrometry, Pharmaqam, Chemistry Department, Université du Québec à Montréal, C.P. 8888 Succ. Centre-Ville, Montréal, Québec, H3C 3P8, Canada.
Background:
Covalent binding by reactive drug metabolites represents a poorly understood cause of drug toxicity. Currently, assessing protein covalent binding usually entails the use of radioactive drug and therefore has limited applicability in drug discovery. Several marketed drugs are known to form reactive metabolites and have been shown to covalently bind to proteins.
Results:
In this article, we describe a new method for the analysis of reactive metabolite-protein binding by MS using a strategy of complete digestion of microsomal proteins into free amino acids. Immobilized pronase was found to be the best method for complete digestion in terms of stability of amino acid modifications as well as minimized spectral background.
Conclusion:
Modified cysteine residues were identified for four tested drug compounds known to form reactive metabolites following in vitro microsomal incubations and accurate mass measurements by LC-MS analysis.
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