Electrospray ionization mass spectrometric analysis of intact cytochrome P450: Identification of tienilic acid adducts to P450 2C9

A general scheme for the purification of baculovirus-expressed cytochrome P 450s (P450s) from the crude insect cell pastes has been designed which rend ers the P450s suitable for analysis by high-performance liquid chromatograp hy (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-M S procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and ra t NADPH cytochrome P450 reductase (P450 reductase). The experimentally dete rmined and predicted (based on the amino acid sequences) molecular masses ( MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, base d on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standa rd deviation of less than 0.09% (based on the charge state distribution). A pplication of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienili c acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 a nd both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the pr esence of glutathione, only native P450 2C9 and the monoadduct were detecte d. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts , a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed .