Mass spectrometry and ion mobility study of poly(ethylene glycol)-based polyurethane oligomers.

RATIONALE: Commercial-grade polymer synthesis is performed via melt polymerization, which leads to polydispersion. The work reported herein provides a synthetic strategy to produce mono-dispersive polyurethane oligomers and an analytical strategy to distinguish these oligomers, providing chemists with the tools necessary to synthesize and identify specific polymer structures that exhibit a desired property. METHODS: Three isomeric poly(ethylene glycol)-polyurethane (PEG-PUR) oligomers were synthesized and analyzed via flow-injection ion mobility mass spectrometry (IM-MS). Each polymer oligomer was injected and run independently via flow injection at 100 µL•min-1 and analyzed in positive ion mode on a drift tube quadrupole time-of-flight (QTOF) instrument. Mobility measurements were determined using a single-field approach. For tandem mass spectrometry (MS/MS) experiments, the sodium-adducted singly charged precursor ion was isolated in the quadrupole and subjected to a range of collision energies. RESULTS: In MS experiments, both +1 and +2 sodium-adducted species were observed for each oligomer at m/z 837.4 and 430.2, respectively. When isolated and fragmented via MS/MS, the +1 precursor yielded distinct product ions for each of the three isomeric oligomers. Fragmentation generally occurred at urethane linkages via 1,3- and 1,5-H shift mechanisms. IM was also used to distinguish the three isomers, with greater IM separation observed for the +2 versus the +1 species. CONCLUSIONS: Mono-disperse PEG-PUR oligomers were synthesized and analyzed. Although the polymeric oligomers analyzed in this study are quite small and structurally simple, this work serves as a model system for the synthesis and structural characterization of larger, more complex block copolymers.

Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures.

We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.

Single Quantum Dot Tracking Illuminates Neuroscience at the Nanoscale.

The use of nanometer-sized semiconductor crystals, known as quantum dots, allows us to directly observe individual biomolecular transactions through a fluorescence microscope. Here, we review the evolution of single quantum dot tracking over the past two decades, highlight key biophysical discoveries facilitated by quantum dots, briefly discuss biochemical and optical implementation strategies for a single quantum dot tracking experiment, and report recent accomplishments of our group at the interface of molecular neuroscience and nanoscience.

Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation.

The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.

4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter.

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.

A fluorescence displacement assay for antidepressant drug discovery based on ligand-conjugated quantum dots.

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.

Biotin tethered homotryptamine derivatives: high affinity probes of the human serotonin transporter (hSERT).

Quantum dot conjugates of compounds capable of inhibiting the serotonin transporter (SERT) could form the basis of fluorescent probes for live cell imaging of membrane bound SERT. Additionally, quantum dot-SERT antagonist conjugates may be amenable to fluorescence-based, high-throughput assays for this transporter. This Letter describes the synthesis of SERT-selective ligands amenable to conjugation to quantum dots via a biotin-streptavidin binding interaction. SERT selectivity and affinity were incorporated into the ligand via a tetrahydropyridine or cyclohexylamine derivative and the affinity of these compounds for SERT was measured by their ability to produce SERT-dependent currents in Xenopus laveis oocytes.

A Novel Biotinylated Homotryptamine Derivative for Quantum Dot Imaging of Serotonin Transporter in Live Cells.

The serotonin transporter (SERT) is the primary target for selective serotonin reuptake inhibitor (SSRI) antidepressants that are thought to exert their therapeutic effects by increasing the synaptic concentration of serotonin. Consequently, probes that can be utilized to study cellular trafficking of SERT are valuable research tools. We have developed a novel ligand (IDT785) that is composed of a SERT antagonist (a tetrahydro pyridyl indole derivative) conjugated to a biotinylated poly ethylene glycol (PEG) via a phenethyl linker. This compound was determined to be biologically active and inhibited SERT-mediated reuptake of IDT307 with the half-maximal inhibitory concentration of 7.2 ± 0.3 µM. We demonstrated that IDT785 enabled quantum dot (QD) labeling of membrane SERT in transfected HEK-293 cultures that could be blocked using the high affinity serotonin reuptake inhibitor paroxetine. Molecular docking studies suggested that IDT785 might be binding to the extracellular vestibule binding site rather than the orthosteric substrate binding site, which could be attributable to the hydrophilicity of the PEG chain and the increased loss of degrees of freedom that would be required to penetrate into the orthosteric binding site. Using IDT785, we were able to study the membrane localization and membrane dynamics of YFP-SERT heterologously expressed in HEK-293 cells and demonstrated that SERT expression was enriched in the membrane edge and in thin cellular protrusions.

GABAC receptor binding of quantum-dot conjugates of variable ligand valency.

Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand.

Ligand-conjugated quantum dots for fast sub-diffraction protein tracking in acute brain slices.

Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.

Quantum dots reveal heterogeneous membrane diffusivity and dynamic surface density polarization of dopamine transporter.

The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.

Single Quantum Dot Imaging Reveals PKCß-Dependent Alterations in Membrane Diffusion and Clustering of an Attention-Deficit Hyperactivity Disorder/Autism/Bipolar Disorder-Associated Dopamine Transporter Variant.

The dopamine transporter (DAT) is a transmembrane protein that terminates dopamine signaling in the brain by driving rapid dopamine reuptake into presynaptic nerve terminals. Several lines of evidence indicate that DAT dysfunction is linked to neuropsychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD), bipolar disorder (BPD), and autism spectrum disorder (ASD). Indeed, individuals with these disorders have been found to express the rare, functional DAT coding variant Val559, which confers anomalous dopamine efflux (ADE) in vitro and in vivo. To elucidate the impact of the DAT Val559 variant on membrane diffusion dynamics, we implemented our antagonist-conjugated quantum dot (QD) labeling approach to monitor the lateral mobility of single particle-labeled transporters in transfected HEK-293 and SK-N-MC cells. Our results demonstrate significantly higher diffusion coefficients of DAT Val559 compared to those of DAT Ala559, effects likely determined by elevated N-terminal transporter phosphorylation. We also provide pharmacological evidence that PKCß-mediated signaling supports enhanced DAT Val559 membrane diffusion rates. Additionally, our results are complimented with diffusion rates of phosphomimicked and phosphorylation-occluded DAT variants. Furthermore, we show DAT Val559 has a lower propensity for membrane clustering, which may be caused by a mutation-derived shift out of membrane microdomains leading to faster lateral membrane diffusion rates. These findings further demonstrate a functional impact of DAT Val559 and suggest that changes in transporter localization and lateral mobility may sustain ADE and contribute to alterations in dopamine signaling underlying multiple neuropsychiatric disorders.

Controlling the reactivity of ampiphilic quantum dots in biological assays through hydrophobic assembly of custom PEG derivatives.

Modifications of the quantum dot (QD) surface are routinely performed via covalent biomolecule attachment, and poly(ethylene glycol) (PEG) derivatization has previously been shown to limit nonspecific cellular interactions of QD probes. Attempts to functionalize ampiphilic QDs (AMP-QDs) with custom PEG derivatives having a hydrophobic terminus resulted in self-assembly of these PEG ligands to the AMP-QD surface in the absence of covalent coupling reagents. We demonstrate, via electrophoretic characterization techniques, that these self-assembled PEG-QDs exhibit improved passivation in biological environments and are less susceptible to unwanted protein adsorption to the QD surface. We highlight the artifactual fluorescent response protein adsorption can cause in biological assays, and discuss considerations for improved small molecule presentation to facilitate specific QD interactions.

Labeling cell-surface proteins via antibody quantum dot streptavidin conjugates.

The quantum dot is a novel fluorescent platform that has the potential to become an alternative to conventional organic dyes used to label biological probes such as antibodies or ligands. Compared to typical fluorescent organic dyes, cadmium selenide/zinc sulfide core-shell nanocrystals, or quantum dots, have greater photostability, resist metabolic and chemical degradation, are nontoxic, and display broad emission and narrow excitation bands. When conjugated to generic adaptor molecules such as streptavidin, quantum dots can be used to label different biotinylated antibodies or ligands without having to customize the quantum dot surface chemistry for each antibody or ligand. In this chapter, we outline the methodology for using streptavidin quantum dots to label biotinylated antibodies that target cell-surface ectodomain proteins on both living and fixed cells.

Peptide-conjugated quantum dots: imaging the angiotensin type 1 receptor in living cells.

Peptide-quantum dot conjugates have been prepared by attaching angiotensin II (Ang II) to cadmium selenide/zinc sulfide core-shell nanocrystals using an 1-[3-(Dimethyamino)propyl]-3-ethylcarbo diimide hydrochloride (EDC) coupling. These conjugates have been used to image angiotensin I-expressing Chinese hamster ovary (CHO) cells in vitro. When CHO cells were incubated with Ang II before incubating with Ang II-conjugated quantum dots, we were able to block the binding of the dots. The Ang II-quantum dot conjugates did not bind to parental cells and showed similar staining patterns when compared with a commercially available Ang II Alexa 488 conjugate.

Single-quantum-dot tracking reveals altered membrane dynamics of an attention-deficit/hyperactivity-disorder-derived dopamine transporter coding variant.

The presynaptic, cocaine- and amphetamine-sensitive dopamine (DA) transporter (DAT, SLC6A3) controls the intensity and duration of synaptic dopamine signals by rapid clearance of DA back into presynaptic nerve terminals. Abnormalities in DAT-mediated DA clearance have been linked to a variety of neuropsychiatric disorders, including addiction, autism, and attention deficit/hyperactivity disorder (ADHD). Membrane trafficking of DAT appears to be an important, albeit incompletely understood, post-translational regulatory mechanism; its dysregulation has been recently proposed as a potential risk determinant of these disorders. In this study, we demonstrate a link between an ADHD-associated DAT mutation (Arg615Cys, R615C) and variation on DAT transporter cell surface dynamics, a combination only previously studied with ensemble biochemical and optical approaches that featured limited spatiotemporal resolution. Here, we utilize high-affinity, DAT-specific antagonist-conjugated quantum dot (QD) probes to establish the dynamic mobility of wild-type and mutant DATs at the plasma membrane of living cells. Single DAT-QD complex trajectory analysis revealed that the DAT 615C variant exhibited increased membrane mobility relative to DAT 615R, with diffusion rates comparable to those observed after lipid raft disruption. This phenomenon was accompanied by a loss of transporter mobilization triggered by amphetamine, a common component of ADHD medications. Together, our data provides the first dynamic imaging of single DAT proteins, providing new insights into the relationship between surface dynamics and trafficking of both wild-type and disease-associated transporters. Our approach should be generalizable to future studies that explore the possibilities of perturbed surface DAT dynamics that may arise as a consequence of genetic alterations, regulatory changes, and drug use that contribute to the etiology or treatment of neuropsychiatric disorders.

Serotonin uptake is largely mediated by platelets versus lymphocytes in peripheral blood cells.

The serotonin transporter (SERT), a primary target for many antidepressants, is expressed in the brain and also in peripheral blood cells. Although platelet SERT function is well accepted, lymphocyte SERT function has not been definitively characterized. Due to their small size, platelets often are found in peripheral blood mononuclear cell preparations aimed at isolating lymphocytes, monocytes, and macrophages. The presence of different cells makes it difficult to assign SERT expression and function to specific cell types. Here, we use flow cytometry and IDT307, a monoamine transporter substrate that fluoresces after uptake into cells, to investigate SERT function in lymphocyte and platelet populations independently, as well as simultaneously without prior isolation. We find that murine lymphocytes exhibit temperature-dependent IDT307 transport but uptake is independent of SERT. Lack of measurable SERT function in lymphocytes was corroborated by chronoamperometry using serotonin as a substrate. When we examined rhesus and human mixed blood cell populations, we found that platelets, and not lymphocytes, were primary contributors to SERT function. Overall, these findings indicate that lymphocyte SERT function is minimal. Moreover, flow cytometry, in conjunction with the fluorescent transporter substrate IDT307, can be widely applied to investigate SERT in platelets from populations of clinical significance.

Quantum dot conjugates of GABA and muscimol: binding to α1ß2γ2 and ρ1 GABA(A) receptors.

GABAA receptors are ligand-gated ion channels that mediate inhibitory synaptic signaling in the CNS. Fluorescent probes with the ability to target these receptors can provide insights into receptor location, distribution and dynamics in live cells, while revealing abnormalities in their distribution and dynamics that could occur in a variety of diseases. We have developed fluorescent probes of GABAA receptors that are composed of a CdSe/ZnS core-shell nanocrystal (quantum dot; qdot) conjugated to pegylated derivatives of the GABA receptor agonists GABA and muscimol (GABA-qdots and muscimol-qdots, respectively). Quantitative fluorescence imaging was used to analyze the binding activity of these conjugates to α1ß2γ2 GABAA and ρ1 GABAA receptors expressed in Xenopus oocytes. The selectivity of these conjugates for α1ß2γ2 GABAA and ρ1 GABAA receptors was determined by their ability to compete with the antagonists bicuculline and methyl-(1,2,3,6-tetrahydropyridin-4-yl)phosphinic acid (TPMPA). Both GABA- and muscimol-qdots exhibited robust binding to both α1ß2γ2 and ρ1 GABAA receptors. At α1ß2γ2 receptors, pretreatment with bicuculline reduced conjugate binding by ≥8-fold on average, an extent far exceeding the reduction produced by TPMPA (~30%). Conversely, at ρ1 receptors, pretreatment with TPMPA inhibited binding by ~10-fold, an extent greatly exceeding the change produced by bicuculline (~50% or less). These results indicate specific binding of muscimol-qdots and GABA-qdots to α1ß2γ2 GABAA and ρ1 GABAA receptors in a manner that preserves the respective pharmacological sensitivities of these receptors to TPMPA and bicuculline, and encourage the use of qdot-conjugated neurotransmitter analogs as labeling agents at GABAA receptors.

A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots.

Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

Biocompatible quantum dots for biological applications.

Semiconductor quantum dots are quickly becoming a critical diagnostic tool for discerning cellular function at the molecular level. Their high brightness, long-lasting, size-tunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biologically oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biologically compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.