Proteomic Quantification of Changes in Abundance of Drug-Metabolizing Enzymes and Drug Transporters in Human Liver Cirrhosis: Different Methods, Similar Outcomes.

Abstract

Model-based assessment of the effects of liver disease on drug pharmacokinetics requires quantification of changes in enzymes and transporters responsible for drug metabolism and disposition. Different proteomic methods are currently used for protein quantification in tissues and in vitro systems, each with specific procedures and requirements. The outcome of quantitative proteomic assays using four different methods (one targeted and three label-free) applied to the same sample set was compared in this study. Three pooled cirrhotic liver microsomal samples corresponding to cirrhosis with nonalcoholic fatty liver disease, biliary disease, or cancer and a control microsomal pool were analyzed using quantification concatemer-based targeted proteomics, the total protein approach (TPA), high three ion intensity (Hi3) approach, and intensity-based absolute quantification (iBAQ) to determine the absolute and relative abundance in disease compared with control. The relative abundance data provided a "disease perturbation factor" (DPF) for each target protein. Absolute and relative abundances generated by standard-based label-free methods (iBAQ and Hi3) showed good agreement with targeted proteomics (limited bias and scatter), but TPA (standard-free method) overestimated absolute abundances by approximately 2-fold. The DPF was consistent between different proteomic methods but varied between enzymes and transporters, indicating discordance of effects of cirrhosis on various metabolism-related proteins. The DPF ranged from no change (e.g., for glucuronosyltransferase-1A6 in nonalcoholic fatty liver disease group) to less than 0.3 (e.g., carboxylesterases-1 in cirrhosis of biliary origin). SIGNIFICANCE STATEMENT: This study demonstrated that relative changes in enzymes and transporters (DPF) are independent of the quantitative proteomic methods used. Standard-based label-free methods, such as high three ion intensity (Hi3) and intensity-based absolute quantification (iBAQ) methods, were less biased and more precise than the total protein approach (TPA) when compared with targeted data. The DPF reconciled differences across proteomic methods observed with absolute levels. Using this approach, differences were revealed in the expression of enzymes/transporters in cirrhosis associated with different etiologies.