One of the central problems in biochemistry in the post-genomic era is the elucidation of functions of proteins, including “orphan” human cytochromes P450 (P450s), when the substrates are unknown. A general strategy for identification of endogenous substrates of P450s in tissue extracts using metabolomic and isotopic labeling approaches is described, involving four main steps: (1) In vitro incubation of a P450 enzyme system with cofactor and tissue extract is done under a mixture of 18O2/16O2 (1:1). (2) LC-MS assay of an organic extract of the reaction mixture is performed. (3) The isotopic labeling products appearing as M/M+2 doublets can be directly identified using the program DoGEX (R. Sanchez-Ponce and F. P. Guengerich, Anal. Chem. 79, 3355-3362, 2007). (4) Characterization of potential candidates is done. Validation of the strategy was established using human P450 7A1 as an initial model to identify its known product, 7α-hydroxycholesterol, in liver extracts. The strategy was then applied to human P450s 1A2, 2C8, and 2C9 in untargeted substrate searches with human liver extracts. A total of seven fatty acids were identified and verified as substrates of these three hepatic P450s. The products were subsequently characterized as hydroxylation and epoxidation derivatives of fatty acids, using GC-MS analysis. Finally, kinetic studies were performed to confirm that the fatty acids are oxidized by P450s 1A2, 2C8, and 2C9. Thus, this strategy has been demonstrated to be useful in identifying reactions in tissue extracts with orphan human P450s.

Chemical derivatization is useful for improving the ionization characteristics of poorly or nonionizable analytes in liquid chromatography-mass spectrometry (LC-MS). Dansyl chloride has been widely used as a derivatizing reagent for fluorescence detection and for facilitating the MS detection of phenols and amines, but not for general alcohols. A new dansylation method for improving the mass spectral sensitivity of unactivated alcohols was developed. The dansylated derivative was formed after incubation of the test compound cholesterol and excess dansyl chloride in CH 2 Cl 2 in the presence of 4-(dimethylamino)-pyridine (DMAP) plus N,Ndiisopropylethylamine at 65 °C for 1 h, with an overall yield of 96%. The versatility of dansylation was investigated by utilizing representative lipid compounds (containing different numbers of hydroxy groups) for dansylation. All dansylated derivatives of the selected compounds were detected by LC-MS/MS in the electrospray ionization (ESI) positive ion mode. Validation of the method was established in terms of the sensitivity, stability, and repeatability of dansylation. The method was then applied to characterizing the P450 7A1 oxidation product (dansylated 7α-hydroxycholesterol) in human liver extracts using an LC-MS metabolomics/isotopic labeling approach (Tang, Z.; Guengerich, F. P. Anal. Chem. 2009, 81, 3071-3078). The dansylated derivative of the product was identified, with the signal increased by 10 3 -fold compared with a previous method (derivatization with succinic anhydride and ESI negative ion MS). Quantitation of testosterone in human liver extracts was also done as an example of the application of the dansylation method. Thus, dansylation is a potential method of modifying many alcohols for detection by fluorescence and LC-MS analysis.

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.

Human cytochrome P450 (P450) 4F11 is still considered an "orphan" because its function is not well characterized. A bacterial expression system was developed for human P450 4F11, producing ~230 nmol P450 from a 3-liter culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic and isotopic labeling approach (Z. Tang et al., Anal. Chem. 81, 3071-3078, 2009). Four fatty acids-palmitic, oleic, arachidonic, and docosahexaenoic-were identified in human liver and verified as substrates of P450 4F11. The products were characterized as ω-hydroxylated fatty acids by gas chromatography-mass spectrometry analysis of their trimethylsilyl derivatives. Kinetic analysis of the oxidation products confirmed that the fatty acids are substrates oxidized by P450 4F11. P450 4F11 also exhibited low activity for some drug Ndemethylation reactions but none for activation of several procarcinogens.