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

• DOI: 10.1124/dmd.112.045054
• Publication Date: 2012-04-14T04:10:17Z
• Authors: Yuichiro Sato; Aiji Miyashita; Takafumi Iwatsubo; Takashi Usui
• Keywords: Hydrolysis; Molecular Sequence Data; Reproducibility of Results; Serum Albumin, Bovine; Peptide Fragments; 03 medical and health sciences; Cytosol; 0302 clinical medicine; Liver; Tandem Mass Spectrometry; Isotope Labeling; Calibration; Microsomes, Liver; Animals; Humans; Cattle; Amino Acid Sequence; Carboxylic Ester Hydrolases; Chromatography, Liquid

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 μM) and oxybutynin (10 μM), whereas CES2 levels correlated strongly with hydrolysis of the CES2 substrate, irinotecan (1 μM), 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.