Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some\ndrugs, and their involvement may lead to drugââ?¬â??drug interaction (DDI) susceptibility when given in combination\nwith drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the\nmagnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification\nof their expression in human intestine, and linking these to data obtained through in vitro experiments.\nA quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem\nmass spectrometry (LCââ?¬â??MS/MS) together with a quantitative concatenation (QconCAT) strategy to\nprovide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase;\nNa/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast\ncancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A\n(Organic Solute Transporter subunit alpha; OST-), in human distal jejunum (n = 3) and distal ileum\n(n = 1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were\noptimised to enable quantification of the proteotypic peptides for each transporter. After harvesting\nenterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were\nsubjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a\ngravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard\n(correlation coefficient, R2 = 0.998) and quantification of all target peptides in a pooled intestinal quality\ncontrol sample (R2 ?\n0.980) was established. The assay was also assessed for within and between-day\nprecision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs,\nover 2 days. The methods were applied to obtain the absolute abundances for all targeted proteins. In\nall samples, Na/K-ATPase, HPT1, P-gp and BCRP were detected above the lower limit of quantitation (i.e.,\n>0.2 fmol/g membrane protein). MRP2 abundance could be quantified in distal jejunum but not in the\ndistal ileum sample. OST- was not detected in 2 out of 3 jejunum samples. This study highlights the\nutility of a QconCAT strategy to quantify absolute transporter abundances in human intestinal tissues.
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