Simultaneous Absolute Protein Quantification of Carboxylesterases 1 and 2 in Human Liver Tissue Fractions using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

ABSTRACT:The aims of this study were to develop a robust method for simultaneous quantification of carboxylesterases (CESs) 1 and 2 and to quantify those absolute protein levels in human liver tissue fractions. Unique peptide fragments of CES1 and CES2 in tryptically digested human liver microsomes (HLMs) and cytosol (HLC) were simultaneously quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using corresponding stable isotope-labeled peptides as internal standards. Bovine serum albumin was used as a blank matrix for the calibration curve samples. Our procedure showed good digestion efficiency, sensitivity, linearity of calibration curve, and reproducibility. The protein levels of CES1 and CES2 in 16 individual HLMs varied 4.7-fold (171-801 pmol/mg) and 3.5-fold (16.3-57.2 pmol/mg), respectively, that are approximately 10 times higher than the expression levels in HLC. The CES1/CES2 level ratio varied substantially from 3.0 to 25, and the correlation between the protein levels of CES1 and CES2 was negative, indicating significant interindividual variability and independence in their expression levels. CES1 levels significantly correlated with hydrolysis of the CES1 substrates, clopidogrel (5 M) and oxybutynin (10 M), whereas CES2 levels correlated strongly with hydrolysis of the CES2 substrate, irinotecan (1 M), indicating that quantified protein levels are highly reliable. This is the first report to demonstrate the absolute protein levels of CESs quantified by LC-MS/MS.

Age-Dependent Absolute Abundance of Hepatic Carboxylesterases (CES1 and CES2) by LC-MS/MS Proteomics: Application to PBPK Modeling of Oseltamivir In Vivo Pharmacokinetics in Infants

“…For example, CES1 abundance in the liver is ;20-to 30-fold higher than the most abundant adult cytochrome P450 enzyme, CYP3A4 (Achour et al, 2014). Interestingly, although the absolute abundance values of CES1 and CES2 presented in this study are consistent with those reported by other LC-MS/MS proteomics studies (Sato et al, 2012;Wang et al, 2016), the values reported by Hines et al (2016) are significantly lower, which might be due to the methodological differences (i.e., LC-MS/MS proteomics versus immunoblotting).…”

Hepatic, Intestinal, Renal, and Plasma Hydrolysis of Prodrugs in Human, Cynomolgus Monkey, Dog, and Rat: Implications for In Vitro–In Vivo Extrapolation of Clearance of Prodrugs

“…However, human CES1 is also expressed in the lung, heart, and kidney, though to a lesser extent (Satoh et al, 2002). This dominant CES1 expression relative to CES2 in the liver was also confirmed by proteomic data, with an average protein expression level of 402 and 30 pmol/mg protein reported in human liver microsomes for CES1 and CES2, respectively (Sato et al, 2012). In addition to the small intestine, human CES2 mRNA data have also been reported for the kidney and to a minor extent for the colon and heart (Satoh et al, 2002;Quinney et al, 2005).…”

Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography–Tandem Mass Spectrometry

“…To select proteotypic peptides that could be digested efficiently, we preliminarily analyzed tryptic digests of rhUGTs using enhanced mass spectrometry scanning, and identified peptide fragments by ProteinPilot (AB SCIEX) according to a published method (data not shown) (Sato et al, 2012a). Proteoptypic peptides identified with the highest confidence values here or reported to yield highly reproducible quantities by others were selected as analytes (Supplemental Table 1) (Fallon et al, 2008;Harbourt et al, 2012;Sato et al, 2012b;Fallon et al, 2013b).…”