Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit critical proteins, elicit immune responses, and cause life threatening adverse drug reactions. The masses of metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification: masses require defining before searching. “Open” search algorithms address this problem. Here, we present Magnum, an open search tool optimized for adduct identification; and Limelight, a web-based data processing package for analysis and visualization of data from all existing search algorithms, incorporating specific tools for sample comparisons and xenobiotic-adduct discovery. We demonstrate, using three drug/protein combinations, our new methods and software tools enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open searching, which until now has had no comprehensive data visualization tools.

Raloxifene, a drug commonly used to treat osteoporosis in postmenopausal women, is a mechanism-based inhibitor of the P450 enzyme CYP3A428. Raloxifene metabolism by CYP3A4 produces several electrophilic species. We incubated CYP3A4 plus P450-reductase to raloxifene in vitro in the presence of NADPH. LC-MS/MS data of exposed and untreated samples were acquired and open modification searches were performed using Magnum. Comparisons of individual raloxifene treated, versus untreated replicates, were performed within Limelight, and a two-tailed test of proportions identified 471 Da as the most significantly enriched mass in the treated sample group. The Skyline files presented here show the XICs of all precursor ions corresponding to amino acid residues identified by Magnum-CWY searches as having a 471 Da modification in ≥ 2 PSMs at a Percolator assigned q ≤ 0.01. Ten peptides not modified by raloxifene were also quantified in treated and untreated samples. These were used for normalization as described in Materials and Methods. See the publication for full details, and specifically Supplementary Note 8.

Rat P450 reductase was expressed and purified as described in DOI: 10.1021/bi2013454. Purified recombinant human CYP3A4 and the liposome stock containing l-α-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), l- α -diloleoyl-sn-glycero-3-phosphocholine (DOPC), and l- α -dilauroyl-sn-glycero-3-phosphoserine (DLPS) (1:1:1, w/w/w per mL) was expressed and purified as described in DOI: 10.1021/acs.biochem.9b01001. Recombinant human CYP3A4 (3 µM) was incubated with 6 µM rat P450 reductase, 2 mM NADPH in the absence and presence of 200 µM raloxifene in buffer containing 20 µg/mL liposomes [DLPC, DOPC, DLPS (1:1:1, w/w/w per mL)], 0.1 mg/mL CHAPS, 3 mM glutathione, 30 MgCl2 and 50 mM Hepes-KOH (pH 7.4). The control group contained no raloxifene. Incubations were done in singlet at 37°C for 1 hour in a total volume of 200 µL and the reaction was started by adding NADPH. Samples were flash-frozen using liquid nitrogen right after the incubation and stored at -80°C until LC-MS analysis.