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.     

MS Binding Assays for the Three Monoamine Transporters Using the Triple Reuptake Inhibitor (1R,3S)‐Indatraline as Native Marker

We herein present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays) for the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT). Using this approach both enantiomers of the triple reuptake inhibitor indatraline as well as its cis‐configured diastereomer were investigated toward hDAT, hNET, and hSERT in saturation experiments. The dissociation rate constants for (1R,3S)‐indatraline binding at hDAT, hNET, and hSERT were determined in kinetic studies. These experiments revealed an allosteric effect of clomipramine on the dissociation of (1R,3S)‐indatraline from hSERT. Finally, a comprehensive set of known monoamine transport inhibitors and substrates was studied in competition experiments at hDAT, hNET, and hSERT, using (1R,3S)‐indatraline as nonlabeled marker. The results are in excellent agreement with those reported for radioligand binding assays. Therefore, the established MS Binding Assays are a promising alternative to the latter for the characterization of new monoamine reuptake inhibitors at DAT, NET, and SERT.     

Quantitative MALDI‐MS Binding Assays: An Alternative to Radiolabeling

Radiolabeling of ligands is still the gold standard in the study of high‐affinity receptor–ligand interactions. In an effort toward safer and simpler alternatives to the use of radioisotopes, we developed a quantitative and highly sensitive matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) method that relies on the use of chemically tagged ligands designed to be specifically detectable when present as traces in complex biological mixtures such as cellular lysates. This innovative technology allows easy, sensitive detection and accurate quantification of analytes at the sub‐nanomolar level. After statistical validation, we were able to perform pharmacological evaluations of G protein‐coupled receptor (V1A‐R)–ligand interactions. Both saturation and competitive binding assays were successfully processed.     

Competitive MS binding assays for dopamine D2 receptors employing spiperone as a native marker

A competitive MS binding assay employing spiperone as a native marker and a porcine striatal membrane fraction as a source for dopamine D2 receptors in a nonvolatile buffer has been established. Binding of the test compounds to the target was monitored by mass-spectrometric quantification of the nonbound marker, spiperone, in the supernatant of the binding samples obtained by centrifugation. A solid-phase extraction procedure was used for separating spiperone from ESI-MS-incompatible supernatant matrix components. Subsequently, the marker was reliably quantified by LC-ESI-MS-MS by using haloperidol as an internal standard. The affinities of the test compounds, the dopamine receptor antagonists (+)-butaclamol, chlorpromazine and (S)-sulpiride obtained from the competitive MS binding assay were verified by corresponding radioligand binding experiments with [3H]spiperone. The results of this study demonstrate that competitive MS binding assays represent a universally applicable alternative to conventional radioligand binding assays.     

(S)- and (R)-fluoxetine as native markers in mass spectrometry (MS) binding assays addressing the serotonin transporter

A recently established and validated LC-ESI-MS/MS method for quantification of fluoxetine was used to implement MS binding assays for the human serotonin transporter (hSERT)-the primary target in the treatment of depression and emotional disorders. As a label-free screening technique, MS binding assays offer the opportunity to perform kinetic, saturation and competition assays using both (S)- and (R)-fluoxetine as native markers. In kinetic experiments, an association rate constant (k(+1) of 0.92±0.17×10(6) M(-1) s(-1) and a dissociation rate constant (k(-1)) of 0.0032±0.0002 s(-1) for (S)-fluoxetine binding to hSERT were determined. Saturation experiments provided K(d) values of 4.4±0.4 nM and 5.2±0.9 nM for (S)- and (R)-fluoxetine, respectively; statistical analysis revealed that the two enantiomers are equipotent. In competitive experiments with (S)-fluoxetine as a marker, K(i) values were obtained for various known inhibitors with a broad range of affinities for hSERT that correlate well with literature data obtained from radioligand binding experiments with [(3)H]imipramine. Additional competitive experiments using (R)-fluoxetine as a marker led to K(i) values for SERT inhibitors that deviate only marginally from those determined using the (S)-enantiomer. No changes in the rank order of affinities occurred, indicating that there is no difference in the binding characteristics of the two enantiomers.     

MS-binding assays: kinetic, saturation, and competitive experiments based on quantitation of bound marker as exemplified by the GABA transporter mGAT1

A new kind of binding assay is described in which the amount of a nonlabeled marker bound to the target is quantified by LC-ESI-MS-MS. This new approach was successfully implemented with nonlabeled NO 711 as marker and the GABA transporter subtype mGAT1 as target. The native marker bound to the target was liberated from the receptor protein by methanol denaturation after filtration. A reliable and sensitive LC-ESI-MS-MS method for the quantitation of NO 711 was developed, and data from mass spectrometric detection were analyzed by nonlinear regression. Kinetic MS-binding experiments yielded values for k+1 and k-1, while in saturation MS-binding experiments, Kd and Bmax values were determined. In competitive MS-binding experiments, Ki values were obtained for various test compounds covering a broad range of affinities for mGAT1. All experiments were performed in 96-well plate format with a filter plate for the separation step which improved the efficiency and throughput of the procedure. The method was validated by classical radioligand-binding experiments with the labeled marker [3H2]NO 711 in parallel. The results obtained from MS-binding experiments were found to be in good agreement with the results of the radioligand-binding assays. The new kind of MS-binding assay presented herein is further adapted to the conventional radioligand-binding assay in that the amount of bound marker is securely quantified. This promises easy implementation in accordance with conventional binding assays without the major drawbacks that are inherent in radioligand or fluorescence binding assays. Therefore, MS-binding assays are a true alternative to classical radioligand-binding assays.     

MS Binding Assays—with MALDI toward High Throughput

A novel type of MS binding assay, a substitute for radioligand binding, in which the quantification of the MS marker is performed by MALDI‐MS–MS (FlashQuant) has been established. Because conventional MS binding assays can only be carried out by LC–ESI‐MS–MS, the use of the FlashQuant system substantially increases the throughput capacity of this method. The study was performed for mGAT1 as a model system. First, a method was developed to quantify NO 711 as a marker for mGAT1 in a range from 208 pM to 16.7 nM using [²H₁₀]NO 711 as an internal standard. On this basis, MS binding assays for mGAT1 could be implemented. Affinity constants determined in both saturation and competition experiments were in excellent agreement with those obtained in MS binding assays based on LC–ESI‐MS–MS quantification. As the MALDI‐MS system takes only a few seconds for quantification per sample, and the whole assay procedure is executed in a 96‐well format, this technique is amenable to high‐throughput screening.     

Indoloquinolizidine–Peptide Hybrids as Multiple Agonists for D1 and D2 Dopamine Receptors

Multiple‐specificity ligands are considered promising pharmacological tools that may show higher efficacy in the treatment of diseases for which the modulation of a single target is therapeutically inadequate. We prepared a set of novel ligands for D₁ and D₂ dopamine receptors by combining two indolo[2,3‐a]quinolizidine scaffolds with various tripeptide moieties. The binding and functional properties of these molecules were determined by radioligand binding studies in brain striatum membranes and by intracellular cAMP production assays in cells expressing different dopamine receptor subtypes. Some indoloquinolizidine–peptide hybrids, mainly with the trans configuration, showed dual agonist activity at both D₁ and D₂ dopamine receptors and may therefore be useful for testing the therapeutic potential of multivalent drugs on these targets.     

Enzymatic Assays Coupled with Mass Spectrometry with or without Embedded Liquid Chromatography

This article reviews monitoring strategies for enzymatic assays coupled with mass spectrometric detection. This coupling has already been shown to be helpful in providing versatile and detailed knowledge about enzyme kinetics. Various available publications address two general approaches. 1) The continuous‐flow setup allows real‐time determination of substrate degradation. Simultaneously, resulting product or potential intermediates can be detected. 2) The online coupled continuous‐flow mixing assay allows the direct coupling of an enzymatic assay to chromatographic separation of complex mixtures. The latest efforts in improving the methodology have been made with regard to miniaturization. This is especially advantageous with regard to reducing costly consumption of chemicals. Finally, these developments are applicable for diverse bioanalytical purposes in the realms of pharmaceutical, biotechnological, food, and environmental research.     

Generation and Screening of Oxime Libraries Addressing the Neuronal GABA Transporter GAT1

The objective of the present study was to transfer the concept of library screening by MS binding assays, so far applied to pseudostatic hydrazine libraries, to static oxime libraries to screen for new potent inhibitors of mGAT1, the most abundant GABA transporter in the central nervous system that represents a validated drug target for the treatment of epilepsy. Library generation was performed by reaction of guvacine derivatives possessing a hydroxylamine functionality with various sets of four aldehydes. After dilution, the libraries were screened by competitive MS binding assays. Deconvolution experiments allowed hits in the most active libraries to be identified, and they were resynthesized for biological evaluation. That way a series of compounds was identified that displayed binding affinities ≥8.00 (pK_i) at mGAT1, one of which was found to be the most potent mGAT1 inhibitor known to date in a functional GABA uptake assay with a pIC₅₀ value of 8.27±0.03.     

Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis

The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which ¹⁵N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O¹⁵NOO⁻), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z = 181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.     

Renal Dopamine Receptors,Oxidative Stress,and Hypertension

Dopamine, which is synthesized in the kidney, independent of renal nerves, plays an important role in the regulation of fluid and electrolyte balance and systemic blood pressure. Lack of any of the five dopamine receptor subtypes (D1R, D2R, D3R, D4R, and D5R) results in hypertension. D1R, D2R, and D5R have been reported to be important in the maintenance of a normal redox balance. In the kidney, the antioxidant effects of these receptors are caused by direct and indirect inhibition of pro-oxidant enzymes, specifically, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase, and stimulation of anti-oxidant enzymes, which can also indirectly inhibit NADPH oxidase activity. Thus, stimulation of the D2R increases the expression of endogenous anti-oxidants, such as Parkinson protein 7 (PARK7 or DJ-1), paraoxonase 2 (PON2), and heme oxygenase 2 (HO-2), all of which can inhibit NADPH oxidase activity. The D5R decreases NADPH oxidase activity, via the inhibition of phospholipase D2, and increases the expression of HO-1, another antioxidant. D1R inhibits NADPH oxidase activity via protein kinase A and protein kinase C cross-talk. In this review, we provide an overview of the protective roles of a specific dopamine receptor subtype on renal oxidative stress, the different mechanisms involved in this effect, and the role of oxidative stress and impairment of dopamine receptor function in the hypertension that arises from the genetic ablation of a specific dopamine receptor gene in mice.     

SPE-GC/MS法检测水体中五氯酚残留

采用固相萃取-气相色谱/质谱法(SPE-GC/MS)研究了温州市水体中的五氯酚(PCP)残留。水样经乙酸酐衍生后用C18固相萃取柱进行富集,10 mL二氯甲烷洗脱,并采用GC/MS法对PCP衍生物五氯苯乙酸酯进行定性定量分析。本实验建立的方法定性定量准确、可靠,适用于水体中PCP的监测,在所有测定的水样中,除温州市区自来水和A、C水库水样以外,其它均有少量PCP检出,最高残留检出量为0.074μg/L。     

基于液相色谱-串联质谱法检测多种食品中的16种合成着色剂

针对多种食品基质(葡萄酒、汽水、黄酒、植物蛋白饮料、乳饮料、花生酱、芝麻酱和糕点)建立了同时测定赤藓红、亮蓝、诱惑红、日落黄、靛蓝、柠檬黄、苏丹红Ⅰ、苏丹红Ⅱ、苏丹红Ⅲ、苏丹红Ⅳ、酸性红1、酸性红4、酸性红8、酸性红14、酸性红88和酸性红97这16种合成着色剂的高效液相色谱-质谱联用分析方法.根据样品种类和所含成分的差别,建立了不同的样品预处理方法,并且通过对提取溶剂的讨论,采用混合溶剂V(甲醇)∶V(乙腈)=2∶3作为提取试剂,比单一溶剂的提取效率更高.萃取液经Kromasil 100-5型色谱柱(150 mm×2.1 mm×5.0 μm)分离,外标法定量.结果表明,16种合成着色剂呈现良好的线性关系(r2 =0.998 9～0.9998),方法的检测限为0.10或0.010 mg/kg,进行低、中、高3个浓度水平添加实验的平均回收率为86.5％ ～97.3％,相对标准偏差＜4.9％(n=6).该方法重现性好,适合于多种食品中上述16种合成着色剂的检测.     

The Separation and Identification of Higher Molecular Weight Fullerenes

A nonaqueous, reversed-phase separation of the fullerenes is described. The bonded-phase, a polymeric octadecyl one, has previously been shown to have a very large separating power for nonplanar polycyclic aromatic hydrocarbons. This work shows that this shape selectivity also is found with the fullerenes, allowing very short retention times while maintaining separation of the C₆₀ and C₇₀ species. Additionally, a fraction containing larger fullerenes was isolated. UV absorbance spectra of these species were obtained. Preliminary spectra of hydrogen chemical ionization mass spectra were also obtained and showed that the fullerenes adsorb large amounts of molecular hydrogen.     

MS-Based Screening of 5-Substituted Nipecotic Acid Derived Hydrazone Libraries as Ligands of the GABA Transporter 1

A screening of compound libraries based on nipecotic acid derivatives with lipophilic residues attached to the scarcely explored 5-position of the core structure was used for the search of new inhibitors of the γ-aminobutyric acid (GABA) transporter 1 (mGAT1). The generated compound libraries, which were based on hydrazone chemistry commonly used in dynamic combinatorial chemistry but rendered pseudostatic, were screened for their binding affinities toward mGAT1 by means of MS Binding Assays. With nipecotic acid derived hydrazone rac- 16 h [rac-(3R,5S)-{5-[(E)-2-{[5-(2-phenylethynyl)thiophen-2-yl]methylidene}hydrazin-1-yl]piperidine-3-carboxylic acid}-sodium chloride (1/2)], one hit was found and evaluated displaying sub-micromolar potency (pK_i=6.62±0.04) and a noncompetitive interaction mode at mGAT1. By bearing a 5-(2-phenylethynyl)thiophen-2-yl residue attached to the 5-position of nipecotic acid via a three-atom spacer, compound rac- 16 h contains a structural moiety so far unprecedented for these kinds of bioactive molecules, and complements novel 5-substituted nipecotic acid derived ligands of mGAT1 revealed in a recently published screening campaign. This new class of ligands, with an inhibition mode distinct from that of benchmark mGAT1 inhibitors, could serve as research tools for investigations of mGAT1-mediated GABA transport.