Characterization of covalent adducts to intact cytochrome P450s by mass spectrometry

This chapter focuses on (1) characterizing MBIs that attach covalently to an active site amino acid and (2) developing an analytical technique that would allow us to accomplish goals in an efficient and accurate manner. To this end, it chooses electrospray ionization (ESI)-mass spectrometry (MS) as method of analysis; this chapter describes use of a rapid, sensitive, and accurate LC/ESI-MS assay to determine the molecular mass of intact P450s prior to and following mechanism-based inactivation by several different compounds. In addition, this technique proved useful for identifying the monoadducts of P450 2A6 and 2B 1 and 8-MOP, 5-MOP, or P, as well as the mono- and diadducts of P450 2C9 and tienilic acid following mechanism-based inactivation. This analytical method was found to be sensitive, rapid, accurate, and one that could be used effectively for the analysis of intact P450s prior to and following mechanism-based inactivation by various compounds. Because it does possess these properties, this procedure should prove useful in high-throughput studies involving covalent adducts of P450s and the characterization of mutant forms.