Covalent binding of reactive metabolites (RMs) to proteins is considered one of the major causes of adverse drug reactions. Consequently, for early drug candidate screening, sensitive methodologies that enable the generation and characterization of reactive metabolites-induced protein adducts in vitro are needed. We have developed a sensitive screening method for drug-protein adducts by using magnetic particles coated with films of metabolic enzymes and target proteins (biocolloid reactors) coupled with capillary liquid chromatography-tandem mass spectrometry. This technique is illustrated by bioactivation of acetaminophen (APAP) into reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which is trapped by covalently binding to the target protein human glutathione S-transferase pi (hGSTP) in the film. Reacted and unreacted hGSTP is then digested by trypsin and subsequently desalted and concentrated using a 10-port switch valve with a trap column (0.5x2mm), followed by analysis by capillary liquid chromatography (Waters capillary LC pumps, 300 um x 15 mm column) coupled to either a Qstar� Elite (AB Sciex) or a Qtrap� 4000 (AB Sciex) mass spectrometer. MASCOT database searches were used to identify the modification site. Multiple reactions monitoring (MRM) was used for more sensitive detection of the target modified peptide. The layer-by-layer coated magnetic particles facilitate simple, fast sample preparation. This method was used to successfully detect the NAPQI-modified peptide ASCLYGQLPK and the characterization of the adduct structure for hGSTP at Cys-47. Therefore, the method was validated as a potential tool for generation and characterization of reactive metabolite-induced protein adducts in vitro and may help to predict adverse drug reactions in early drug development.

Generation and characterization of protein�metabolite adducts using magnetic biocolloid reactors and capillary liquid chromatography�mass spectrometry

246th National Meeting (2013)

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