Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC-MS/MS.

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-

SUBMITTER: Jiang D 

PROVIDER: S-EPMC7509849 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature