Abstract
A new 96-well plate methodology for fast, enzyme-multiplexed screening for metabolite-protein adducts was developed. Magnetic beads coated with metabolic enzymes were used to make potentially reactive metabolites that can react with test protein in the wells, followed by sample workup in multiple 96-well filter plates for LC-MS/MS analysis. Incorporation of human microsomes from multiple organs and selected supersomes of single cytochrome P450 (cyt P450) enzymes on the magnetic beads provided a broad spectrum of metabolic enzymes. The reacted protein was then isolated, denatured, reduced, alkylated, and digested, and peptides were collected in a sequence of 96-well filter plates for analysis. Method performance was evaluated by trapping acetaminophen reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) with human glutathione S-transferase pi (hGSTP), human serum albumin (HSA), and bovine serum albumin (BSA) as model target proteins. Relative amounts of acetaminophen metabolite and hGSTP adducts were compared with 10 different cyt P450 enzymes. Human liver microsomes and CYP1A2 supersomes showed the highest bioactivation rate for adduct formation, in which all four cysteines of hGSTP reacted with NAPQI. Eight cysteines of HSA and four cysteines of BSA have been detected to react with NAPQI. This method has the potential for fast multienzyme protein adduct screening with high efficiency and accuracy.