Efficient protein quantification in drug metabolism and pharmacokinetics with an accelerated proteomic workflow

Quantifying proteins involved in the absorption, distribution, metabolism, and excretion (ADME) of drugs is essential to improve understanding of their disposition and pharmacokinetics. Proteomics, because of its great versatility, is a widely used approach for protein analysis. However, existing protocols face challenges, such as poor peptide identification in liquid chromatography with tandem mass spectrometry under multiple reaction monitoring mode as well as the time- and labor-intensive nature of detergent-engaged workflows. In this study, we compared and evaluated several targeted ADME proteomic methods, including a novel approach called Fast Surfactant-Treated (FAST). Using FAST in ADME proteome analysis of primary human hepatocytes revealed that most proteins, especially membrane proteins, were efficiently solubilized and digested, with the ionic detergent sodium deoxycholate and rapidly removed during preparation by the incorporation of a centrifugation step following acetonitrile precipitation. Compared with the traditional proteomic workflow involving dithiothreitol reduction and iodoacetamide alkylation, FAST achieved an approximately 4-fold increase in cytochrome P450 and UDP-glucuronosyltransferases quantification and 5-fold increase in transporters, based on endogenous tryptic peptide signals. For specific proteins such as CYP2J2, organic anion transporter, and organic anion transporting polypeptide 1B1, FAST generated peptide quantification peaks with significantly higher signal-to-noise ratios and in a shorter amount of sample processing time. We then further validated the FAST proteomics workflow using the pregnane X receptor agonist rifampicin in human hepatocytes, which revealed that CYP3A4 protein levels were induced to a similar extent as observed in the CYP3A midazolam-1'-hydroxylase activity assay. Altogether, these results suggest that FAST proteomics is a robust, efficient, and versatile method for ADME bioanalysis. SIGNIFICANCE STATEMENT: Quantifying absorption, distribution, metabolism, and excretion (ADME) proteins from in vitro matrices remains a challenge, particularly when speed and efficiency are critical. By incorporating sodium deoxycholate detergent into the ADME proteome sample preparation workflow, we developed a methodology called Fast Surfactant-Treated proteomics. This approach enabled more efficient quantification of drug-metabolizing enzymes and membrane transporters, offering a streamlined protocol with reduced bench time.