Characterizing Proteins of Unknown Function: Orphan Cytochrome P450 Enzymes as a Paradigm

Use of the program DoGEX in the analysis of substrates of P450 7A1 with human liver extracts. A reconstituted P450 7A1 system was incubated with NADPH and human liver extract under a 1:1 (v/v) atmosphere of ¹⁸O₂and ¹⁶O₂. LC-MS analysis of an organic extract (derivatized with succinic anhydride) yielded the total ion current trace (part A). Analysis with DoGEX software yielded the profile shown in part B, where a green band signifies a ratio of M and M+2 peaks near 0.95, the target. The positive region of the spectrum is expanded in part C and shown in three dimensions in part D, with a m/z 603/601 doublet at t_R 7 min identified. Confirmation as 7α-hydroxycholesterol (succinic ester) was performed with standard material. See (33).