Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters

In this work, we present label-free, mass-spectrometry-based binding assays (MS Binding Assays), targeting the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) in simultaneous binding experiments. Using a validated LC-ESI-MS/MS method for quantification of the selective dopamine transporter inhibitor (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol ((R,R)-D-84), the selective norepinephrine transporter inhibitor (S,S)-reboxetine, and the selective serotonin reuptake inhibitor (S)-citalopram, binding affinities at the three monoamine transporters could be characterized simultaneously in a single binding experiment. The performed simultaneous saturation and competition experiments yielded results that are in good accordance with those determined in MS Binding Assays addressing the monoamine transporters individually. The results obtained from this study underscore the potential of MS Binding Assays for simultaneous affinity determination at different targets, which is difficult to accomplish with conventional radioligand binding assays.