title: 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.
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford M6 8HD, UK; Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield S2 4SU, UK. Electronic address: matthew.harwood@certara.com.
authors: M D
authors: MD
authors: Harwood
authors: Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
authors: B
authors: B
authors: Achour
authors: Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
authors: M R
authors: MR
authors: Russell
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford M6 8HD, UK.
authors: G L
authors: GL
authors: Carlson
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford M6 8HD, UK.
authors: G
authors: G
authors: Warhurst
authors: Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield S2 4SU, UK; Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
authors: A
authors: A
authors: Rostami-Hodjegan
mesh_terms: D002138:Calibration; D002462:Cell Membrane / Q000737:chemistry; D002853:Chromatography, Liquid / Q000592:standards; D020895:Enterocytes / Q000737:chemistry; D006801:Humans; D007082:Ileum / Q000737:chemistry* / Q000166:cytology; D007583:Jejunum / Q000737:chemistry* / Q000166:cytology; D016014:Linear Models; D026901:Membrane Transport Proteins / Q000032:analysis*; D000089762:Multidrug Resistance-Associated Protein 2; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: C000715087:ABCC2 protein, human; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2
keywords: Absolute abundance; Human intestine; QconCAT; Quantitative targeted absolute proteomics; Transporter proteins