A protein standard addition framework for absolute quantification of drug metabolizing enzymes

Accurate quantification of absorption, distribution, metabolism and elimination (ADME) proteins in complex human-derived matrices remains technically challenging. Although targeted bottom-up proteomic approaches have improved specificity and sensitivity relative to immunometric methods, peptide level absolute quantification (AQUA) remains confounded by peptide-dependent proteolytic recovery, limiting confidence in protein abundance estimates. Here, we describe AQUA-ADME, a protein level absolute quantification strategy that integrates protein standard addition with our previously described FAst Surfactant-Treated -enabled liquid chromatography-multiple reaction monitoring workflow to address peptide-dependent digestion bias. By spiking known amounts of purified recombinant human CYP3A4 into native human intestinal and hepatic microsomes prior to digestion, AQUA-ADME enforces matched proteolytic behavior between endogenous CYP3A4 and exogenously spiked recombinant human CYP3A4 within the same biological matrix, while stable isotope-labeled peptides correct for ionization variability. Using 4 structurally and spatially distinct CYP3A4 tryptic peptides, AQUA-ADME yielded highly concordant CYP3A4 abundance estimates within each microsomal pool (<2-fold variation across peptides), in contrast to the wide variability observed using conventional peptide-centric AQUA workflows. Normalization of microsomal CYP3A4-mediated midazolam 1'-hydroxylation and testosterone 6β-hydroxylation rates to AQUA-ADME-derived protein abundance collapsed apparent differences in turnover numbers and specificity constants between intestinal and hepatic microsomes, supporting enzyme abundance as the dominant driver of tissue-specific metabolic capacity. Collectively, these findings demonstrate that AQUA-ADME provides a simple, accessible, and mechanistically grounded approach for absolute protein quantification, strengthening the quantitative foundation for in vitro-in vivo extrapolation and physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Reliable absolute proteomic quantification is essential for confident interpretation of enzyme-normalized kinetic parameters and translational pharmacokinetic modeling. This study introduces AQUA-ADME, a protein standard addition approach that addresses peptide-dependent digestion bias inherent to peptide-centric proteomics. By enforcing matched digestion between endogenous and reference protein, AQUA-ADME yields coherent, protein level abundance estimates and improves confidence in derived turnover numbers. AQUA-ADME provides a mechanistically grounded framework to strengthen quantitative ADME analyses supporting in vitro-in vivo extrapolation and physiologically based pharmacokinetic applications.