Ultrasensitive Quantification of Drug-metabolizing Enzymes and Transporters in Small Sample Volume by Microflow LC-MS/MS

Abstract

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are broadly applicable to the characterization of in vitro and in vivo models, in vitro to in vivo extrapolation (IVIVE), and interindividual variability prediction. However, the emerging need of DMET quantification in small sample volumes such as organ-on a chip effluent, organoids, and biopsies requires ultrasensitive protein quantification methods. We present an ultrasensitive method that relies on an optimized sample preparation approach involving acetone precipitation coupled with a microflow-based liquid chromatography-tandem mass spectrometry (µLC-MS/MS) for the DMET quantification using limited sample volume or protein concentration, i.e., liver tissues (1-100 mg), hepatocyte counts (~4000 to 1 million cells), and microsomal protein concentration (0.01-1 mg/ml). The method was applied to quantify DMETs in differential tissue S9 fractions (liver, intestine, kidney, lung, and heart) and cryopreserved human intestinal mucosa (i.e., CHIM). The method successfully quantified >75% of the target DMETs in the trypsin digests of 1 mg tissue homogenate, 15,000 hepatocytes, and 0.06 mg/ml microsomal protein concentration. The precision of DMET quantification measured as the coefficient of variation across different tissue weights, cell counts, or microsomal protein concentration was within 30%. The method confirmed significant extrahepatic abundance of non-cytochrome P450 enzymes such as dihydropyridine dehydrogenase (DPYD), epoxide hydrolases (EPXs), arylacetamide deacetylase (AADAC), paraoxonases (PONs), and glutathione S-transferases (GSTs). The ultrasensitive method developed here is applicable to characterize emerging miniaturized in vitro models and small volume biopsies. In addition, the differential tissue abundance data of the understudied DMETs will be important for physiologically-based pharmacokinetic (PBPK) modeling of drugs.

Keywords: Drug-metabolizing enzymes, and transporters; In vitro to in vivo extrapolation; Liquid chromatography-mass spectroscopy; Physiology-based pharmacokinetic modeling; Proteomics.

Figure 1:

Sample preparation workflow for the quantification of DMETs in samples isolated from six different human liver tissue weights ranging 1–100 mg (A), 3900 to 1 million cells/ml human hepatocytes (B) HLM and HIM concentrations ranging from 10 to 1000 μg/ml (C). For tissue samples, membrane and non-membrane fractions were isolated and a uniform final protein concentration of 1 mg/ml was used for further analysis. For hepatocytes, the cell lysate was directly used for the further processing, whereas HLM and HIM samples were diluted using human serum albumin (HSA) to keep the total protein concentration at 1 mg/ml. All samples were denatured, reduced, alkylated, and desalted using acetone prior to trypsin digestion. The analysis was performed using μLC-MS/MS at a mobile phase flow rate of 3 μl/min. Data analysis was carried out by Skyline.

Figure 2:

Protein abundance of CYPs, UGTs, and other non-CYPs in samples (membrane or non-membrane fractions) isolated from differential liver tissue weights ranging from 1–100 mg. CYPs, UGTs, and FMO3 were quantified in the membrane fractions, whereas CESs and AO were quantified in the non-membrane fraction. Data are presented as mean and standard deviation of the protein abundance values (pmol/mg protein) calculated across six tissue weights.

Figure 3:

Protein abundance (pmol) of CYPs (A) UGTs (B), and other non-CYPs (C) in 24–6250 cells human hepatocytes on-column. Cell-count normalized fractional abundance (%) abundance of CYPs (D), UGTs (E), and other non-CYPs (F) calculated by dividing the abundance value by the on-column hepatocyte cell count. Fractional abundance (%) of CYPs (G), UGTs (H), and other non-CYPs (I) reported as means and standard deviations of the cell-count normalized values. CYP3A4, CYP2E1, CYP2B6, CES1, and UGT1A3 were not detected in low cell count. The figure insets are zoomed data for low abundance proteins.

Figure 4:

Protein abundance in human liver (H) and intestine (I) microsomes. Protein abundance (pmol/mg) of CYPs (A) UGTs (B), and other non-CYPs (C) in different microsomal on-column protein amount ranging from 5–333 ng. Protein amount normalized fractional abundance (%) of CYPs (D), UGTs (E), and other non-CYPs (F). Fractional abundance (%) of CYPs (G), UGTs (H), and other non-CYPs (I) reported as means and standard deviations of the protein normalized abundance values. The protein amount normalized abundance was calculated by dividing the abundance value by the on-column microsomal protein amount. The figure insets are zoomed data for low abundance proteins.