Protein Abundance of Clinically Relevant Drug-Metabolizing Hydrolases in Human Liver and Intestine by Targeted versus Global Proteomics Approaches: A Comparative Study

While cytochrome P450 enzymes and UDP-glucuronosyltransferases play predominant roles in drug metabolism, hydrolases are emerging as key players in the metabolism of both small molecules and antibody-drug conjugates or peptide–oligonucleotide conjugates. Despite their importance, the protein levels of hydrolases across tissues and their inter-individual variability remain poorly understood. Although targeted proteomics can provide high selectivity and precision in quantifying small numbers of proteins, total protein approach (TPA)-based proteomics is emerging as a superior approach for multiplexed quantification of proteins. We performed a head-to-head comparison of targeted proteomics and TPA-based proteomics for quantifying 12 clinically relevant hydrolases in human liver and intestinal S9 fractions ( N = 5 each). TPA-based global proteomics offered higher precision (coefficient of variation <20%), comparable sensitivity, along with its inherent advantage of a greater protein coverage than targeted proteomics. TPA data revealed the following order of protein abundance for target proteins: CES1> EPHX1 > CES2 > BPHL > PON3 > PON1 ∼ AADAC > CTSA > DPP4 in the liver and CES2 > ADA > DPEP1 ∼ AADAC ∼ EPHX1 > ALPI > DPP4 > CTSA > BPHL ∼ CES1 in the intestine. This study highlights the utility of TPA-based global proteomics for characterizing differential tissue abundance of hydrolases and their inter-individual variability.