<i>In vitro</i> studies with two human organic anion transporters: OAT2 and OAT7

<p>1.Penciclovir, ganciclovir, creatinine, <i>para</i>-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7.</p> <p>2.For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS.</p> <p>3.Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC₅₀ of 17, 22, 23, 24, 35 and 586 μM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC₅₀ = 27 µM; >90% inhibition) by plated human hepatocytes (PHH).</p> <p>4.It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.</p

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS¹ level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS¹ peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied