Application of an LC–MS/MS method for the simultaneous quantification of human intestinal transporter proteins absolute abundance using a QconCAT technique

Abstract

Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments. A quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) together with a quantitative concatenation (QconCAT) strategy to provide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase; Na/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast cancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A (Organic Solute Transporter subunit alpha; OST-α), in human distal jejunum (n=3) and distal ileum (n=1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were optimised to enable quantification of the proteotypic peptides for each transporter. After harvesting enterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were subjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a gravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard (correlation coefficient, R(2)=0.998) and quantification of all target peptides in a pooled intestinal quality control sample (R(2)≥ 0.980) was established. The assay was also assessed for within and between-day precision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs, over 2 days. The methods were applied to obtain the absolute abundances for all targeted proteins. In all samples, Na/K-ATPase, HPT1, P-gp and BCRP were detected above the lower limit of quantitation (i.e., >0.2 fmol/μg membrane protein). MRP2 abundance could be quantified in distal jejunum but not in the distal ileum sample. OST-α was not detected in 2 out of 3 jejunum samples. This study highlights the utility of a QconCAT strategy to quantify absolute transporter abundances in human intestinal tissues.

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

“…(3,37,46); the overall trends suggest substantial inter-individual variabilities in the expression of drug transporters, consistent with limited available functional activity data (37). Quantitative proteomic transporter abundance data are available for human organs such as intestine, liver and brain (95)(96)(97), as well as rat kidney (98). Data for human kidney have recently been published; of the solute carrier transporters (SLC), MATE1 and OAT3 were the most abundant (10.8 and 9.7 pmol/mg microsomal protein, respectively), whereas Pgp/MDR1 was the most abundant ATP-binding cassette (ABC) transporter (4.45 pmol/mg microsomal protein) (22).…”

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells

“…Total membrane preparations were isolated by differential centrifugation after eluting human enterocytes by a calcium chelation protocol (Harwood et al, 2015). Caco-2 cell monolayers total membrane preparations were obtained after overnight lysing by differential centrifugation (Russell et al, 2013).…”

“…However, to our knowledge, studies comparing the quantification of absolute transporter-protein abundances and the subsequent generation of REFs between laboratories for the same samples have not been reported in the literature yet. While not within the scope of this article, the impact of those laboratory-specific REF values on predicted pharmacokinetic outcomes in an IVIVE-PBPK model are provided in an accompanying study (Harwood et al, 2015). The aim of this study was to compare the absolute transporter abundances for three transporter proteins; Na/K-ATPase, P-gp, and BCRP, in matched Caco-2 cell and human intestinal total membrane fractions, quantified by two independent laboratories, the University of Manchester (UoM), Manchester, UK, and Bertin Pharma (BPh), Orleans, France, with each laboratory using different QTAP workflows.…”

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…Several studies published recently reported different proteomic methodologies driven by advances in LC-MS technology (Fallon et al, 2008;Ohtsuki et al, 2012;Achour et al, 2014a;Vildhede et al, 2014;Harwood et al, 2015;Fallon et al, 2016). These methodologies focused on obtaining expression values from a range of mammalian tissues/organs (e.g., liver, intestine, kidneys) and in vitro systems (e.g., hepatocytes, Caco-2 cell lines), with a view to providing systems data for in vitro-in vivo extrapolation (IVIVE) of pharmacokinetic profiles using computerized physiologically based pharmacokinetic (PBPK) models (Rostami-Hodjegan, 2012;Bosgra et al, 2014).…”

“…Sample preparation can also contribute to this variability, with proteins either quantified directly in whole cell/tissue lysate (Weiß et al, 2015;Wi sniewski et al, 2016) or in enriched subcellular fractions Gröer et al, 2013). In addition, differences in proteolytic strategies, efficiency of protein and peptide recovery and LC-MS analysis of peptides have also been suggested to introduce bias into abundance measurements (Chiva et al, 2014;Harwood et al, 2015). Other studies suggested that different analytical methods used in the same laboratory setting can generate reasonably consistent measurements (Qiu et al, 2013;.…”