A family of QconCATs (Quantification conCATemers) for the quantification of human pharmacological target proteins
Document Type
Article
Date of Original Version
6-15-2022
Abstract
We have developed a family of QconCAT standards for the absolute quantification of pharmacological target proteins in a variety of human tissues. The QconCATs consist of concatenated proteotypic peptides, are designed in silico, and expressed in E. coli in media enriched with [13C6] arginine and [13C6] lysine to generate stable isotope-labeled multiplexed absolute quantification standards. The so-called MetCAT (used to quantify cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes), the liver TransCAT (used to quantify plasma-membrane drug transporters) and the brain TransCAT (used to quantify transporters expressed in the blood-brain barrier) were previously reported. We now report new QconCATs for the quantification of non-UGT non-CYP drug metabolizing enzymes (NuncCAT) and receptor tyrosine kinases (KinCAT). We have also redesigned the liver TransCAT, replacing problematic peptides and the N-terminal tag, for better characterization and expression. All these QconCATs showed high purity, high labelling efficiency with stable 13C isotope (>95%), and high sequence coverage (>88%). They represent a close-knit family of standards for quantifying pharmacokinetic targets, together with a more distant cousin, the KinCAT, used to quantify pharmacodynamic targets. Significance: Multiplexed determination of absolute protein abundances using quantitative conCATemers (QconCATs) has already been successfully demonstrated in different human tissues. We have previously reported two QconCATs; MetCAT and TransCAT, for the quantification of key enzymes (cytochrome P450 enzymes (CYP) and glucuronosyltransferases (UGT)) and drug transporters. To build on these reports, application of the QconCAT methodology for the determination of non-UGT non-CYP enzymes and receptor tyrosine kinases (RTKs) in human tissue is reported here. This report focuses on development and characterization of two QconCAT constructs for the quantification of 24 enzymes and 21 RTKs. We demonstrate that the developed QconCATs have high purity, high incorporation efficiency and low peptide miscleavage upon proteolysis. Application of these QconCATs for reliable quantification of target proteins was achieved in human liver.
Publication Title, e.g., Journal
Journal of Proteomics
Volume
261
Citation/Publisher Attribution
Vasilogianni, Areti-Maria, Eman El-Khateeb, Brahim Achour, Sarah Alrubia, Amin Rostami-Hodjegan, Jill Barber, and Zubida M. Al-Majdoub. "A family of QconCATs (Quantification conCATemers) for the quantification of human pharmacological target proteins." Journal of Proteomics 261, (2022). doi: 10.1016/j.jprot.2022.104572.