JIOMICS

JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62
JOURNAL OF INTEGRATED OMICS
Journal of Integrated Omics
A METHODOLOGICAL JOURNAL
HTTP://WWW.JIOMICS.COM
Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015)
Quantitative proteomics in human drug metabolism
Brahim Achour 1 , Matthew R. Russell 1 , Francesco Lanucara 2 , Amin Rostami-Hodjegan 1,3 , Jill Barber* 1
1
Manchester Pharmacy School, University of Manchester, Stopford Building, Oxford Road, Manchester, UK, M13 9PT. 2 Waters Corporation,
Stamford Avenue, Altrincham Road, Wilmslow, UK, SK9 4AX. 3 Simcyp limited (a Certara Company), Blades Enterprise Centre, John Street,
Sheffield, UK, S2 4SU. *Corresponding author: jill.barber@manchester.ac.uk
Available Online: 31 December 2015
Abstract
Purpose: The aim of this study was to develop reproducible, accurate methods for determining the concentrations of enzymes and transporters
involved in human drug metabolism.
Experimental description: We have developed QconCAT-based LC-MS/MS methodology for quantifying the cytochrome P450 and UGT
enzymes and the drug transporters in human tissue. The QconCATs (artificial proteins consisting of labelled standard peptides, released by
proteolysis) initially did not express in E. coli and methods for expressing recalcitrant QconCATs were developed as a part of the project. Specifically,
we fused the two QconCATs (MetCAT and TransCAT which quantify enzymes and transporters respectively) to proteins with known
characteristics. The MetCAT was fused to a QconCAT protein, known to express in inclusion bodies in high yield, whereas the TransCAT was
fused to a soluble construct and expressed in cytosol.
The MetCAT has now been used to quantify the enzymes in a panel of 24 livers, and the TransCAT has been used to quantify transporters in four intestinal
samples. We have also analysed three liver samples using label-free methodology.
Results: Cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes are responsible for much of the metabolism of drugs
in the liver, intestine and other tissues. There is, however, substantial inter-individual variability in their concentrations, as well as variation
between groups of individuals (ethnic groups, age groups). Transporter proteins are responsible for further variations in how a given dose of a
drug may lead to different plasma and tissues in different people and groups.
Our results show wide variation in a population between the levels of the different enzymes, for example CYP3A4, which metabolizes many
important drugs, is present with a range 10.4–262.1 pmol mg -1 protein. We determined several correlations between enzymes, including a
positive correlation between levels of CYP3A4 and CYP2B6.
Most recently, label-free quantification has been shown to give broadly comparable results and, in addition, to give quantification data on other
proteins in the samples.
Conclusions: Personalized medicine is crucially dependent on an understanding of the concentrations of drug-metabolizing enzymes and
transporters in human tissue. Quantitative LC-MS/MS is a powerful tool for understanding these enzymes and transporters, and QconCAT
and label-free quantification methods deliver complementary but consistent information.
Keywords: QconCAT, label-free, quantification (quantitation), cytochrome P450, UGTs.
Acknowledgements: Mass spectrometry was performed at the Michael Barber Centre for Mass Spectrometry, University of Manchester.
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