Biomedical subjects

Stanislav S Rubakhin

Publications and source records attributed to Stanislav S Rubakhin.

At least 19 recordsLinked to original sources

PubMed2006

Profiling signaling peptides in single mammalian cells using mass spectrometry.

The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and nonenzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than 15 peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and postsource decay sequencing allows identification of 14 of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types.

Animals↗

PubMed2006

Massively parallel sample preparation for the MALDI MS analyses of tissues.

Investigation of the peptidome of the nervous system containing large, often easily identifiable neurons has greatly benefited from single-cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and has led to the discovery of hundreds of novel cell-to-cell signaling peptides. By combining new sample preparation methods and established protocols for bioanalytical mass spectrometry, a high-throughput, small-volume approach is created that allows the study of the peptidome of a variety of nervous systems. Specifically, approximately single-cell-sized samples are rapidly prepared from thin tissue slices by adhering the tissue section to a glass bead array that is anchored to a stretchable membrane. Stretching the membrane fragments the tissue slice into thousands of individual samples, their dimensions predominately governed by the size of the individual glass beads. Application of MALDI matrix, followed by the repeated condensation of liquid microdroplets on the fragmented tissue, allows for maximal analyte extraction and incorporation into MALDI matrix crystals. During extraction, analyte migration between the pieces of tissue on separate beads is prevented by the underlying hydrophobic substrate and by controlling the size of the condensation droplets. The procedure, while general in nature, may be tailored to the needs of a variety of analyses, producing mass spectra equivalent to those acquired from single-cell samples.

Animals↗

PubMed2006

Measuring nitric oxide in single neurons by capillary electrophoresis with laser-induced fluorescence: use of ascorbate oxidase in diaminofluorescein measurements.

As a family of novel fluorescent indicators for nitric oxide (NO), the diaminofluoresceins (DAFs) have allowed real-time measurement of neuronal NO, an important gaseous neurotransmitter. However, the measurement of NO by the most commonly used NO sensor, 4,5-diaminofluorescein (DAF-2), is altered by two processes: the interaction of DAF-2 with intracellular dehydroascorbic acid (DHA) and the impact of ascorbic acid (AA) on the levels of N2O3, the intermediate product of the oxidation of NO that reacts with DAF-2. Similar AA/DHA effects are observed with other DAF probes, including DAF-FM and DAR-4M. To overcome these limitations, we use a specific enzymatic reaction to eliminate the confounding effect of AA on DAF quantitation of NO and then use capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to distinguish the various reaction products. First, the enzyme ascorbate oxidase (AO) is used to catalyze the oxidation of AA to DHA. Next, CE-LIF separates the fluorescent products of the reaction of DAF-2 with NO and DHA. Control experiments, including standard mixtures and single neurons with added NO donor, successfully demonstrate the utility of this approach. This protocol is further tested with homogenates of the mouth area from the sea slug Aplysia californica, previously shown to be NO-positive, and individual nitric oxide synthase-containing buccal neurons from the freshwater snail, Lymnaea stagnalis. In each case, significant amounts of NO are detected. This AO DAF methodology is specific, effective, simple, and allows NO to be measured in single cells without detectable interference from other compounds.

Animals↗

PubMed2006

D-Aspartate as a putative cell-cell signaling molecule in the Aplysia californica central nervous system.

The content, synthesis and transport of D-aspartate (D-Asp) in the CNS of Aplysia californica is investigated using capillary electrophoresis (CE) with both laser-induced fluorescence and radionuclide detection. Millimolar concentrations of D-Asp are found in various regions of the CNS. In the cerebral ganglion, three adjacent neuronal clusters have reproducibly different D-Asp levels; for example, in the F- and C-clusters, up to 85% of the free Asp is present in the D-form. Heterogeneous distribution of D-Asp is also found in the individual identified neurons tested, including the optical ganglion top-layer neurons, metacerebral cells, R2 neurons, and F-, C- and G-cluster neurons. The F-cluster neurons have the highest percentage of D-Asp (approximately 58% of the total Asp), whereas the lowest value of approximately 8% is found in R2 neurons. In pulse-chase experiments with radiolabeled D-Asp, followed by CE with radionuclide detection, the synthesis of D-Asp from L-aspartate (L-Asp) is confirmed. Is D-Asp in the soma, or is it transported to distantly located release sites? D-Asp is clearly detected in the major nerves of A. californica, including the pleuroabdominal and cerebrobuccal connectives and the anterior tentacular nerves, suggesting it is transported long distances. In addition, both D-Asp and L-Asp are transported in the pleuroabdominal connectives in a colchicine-dependent manner, whereas several other amino acids are not. Finally, d-Asp produces electrophysiological effects similar to those induced by L-Asp. These data are consistent with an active role for D-Asp in cell-to-cell communication.

Animals↗

PubMed2006

Serotonin catabolism and the formation and fate of 5-hydroxyindole thiazolidine carboxylic acid.

Serotonin (5-HT) functions as a neurotransmitter and neuromodulator in both the central and enteric nervous systems of mammals. The dynamic degradation of 5-HT metabolites in 5-HT-containing nervous system tissues is monitored by capillary electrophoresis with wavelength-resolved laser-induced native fluorescence detection in an effort to investigate known and novel 5-HT catabolic pathways. Tissue samples from wild type mice, genetically altered mice, Long Evans rats, and cultured differentiated rat pheochromocytoma PC-12 cells, are analyzed before and after incubation with excess 5-HT. From these experiments, several new compounds are detected. One metabolite, identified as 5-hydroxyindole thiazoladine carboxylic acid (5-HITCA), has been selected for further study. In 5-HT-incubated central and enteric nervous system tissue samples and differentiated PC-12 cells, 5-HITCA forms at levels equivalent to 5-hydroxyindole acetic acid, via a condensation reaction between L-cysteine and 5-hydroxyindole acetaldehyde. In the enteric nervous system, 5-HITCA is detected without the addition of 5-HT. The levels of L-cysteine and homocysteine in rat brain mitochondria are measured between 80 and 140 microm and 1.9 and 3.4 microm, respectively, demonstrating that 5-HITCA can be formed using available, free L-cysteine in these tissues. The lack of significant accumulation of 5-HITCA in the central and enteric nervous systems, along with data showing the degradation of 5-HITCA into 5-hydroxyindole acetaldehyde, suggests that an equilibrium coupled to the enzyme, aldehyde dehydrogenase type 2, prevents the accumulation of 5-HITCA. Even so, the formation of 5-HITCA represents a catabolic pathway of 5-HT that can affect the levels of 5-HT-derived compounds in the body.

Animals↗

PubMed2006

Identification and characterization of homologues of vertebrate beta-thymosin in the marine mollusk Aplysia californica.

The beta-thymosins have been known as actin-sequestering proteins, but now are recognized as molecules with multiple and diverse intracellular and extracellular functions. Two closely related proteins, beta-thymosin(His) and beta-thymosin(Gln), have been de novo sequenced by top-down mass spectrometry in the common neurobiology model, Aplysia californica. As determined by nanoelectrospray quadrupole-enhanced Fourier-Transform mass spectrometry with collisionally activated and electron-capture dissociations, both of these Aplysia beta-thymosins are acetylated and differ by a single residue in the central actin-binding domain. Profiling of individual cells and tissue by matrix-assisted laser desorption/ionization mass spectrometry reveals that these proteins are widely expressed in the Aplysia central nervous system, including in individual identified neurons, neuronal clusters, nerves and connective tissues. Newly identified beta-thymosin(His) and beta-thymosin(Gln) are also detected by mass spectrometry in hemolymph, and in releasates collected from whole ganglia. When applied exogenously, beta-thymosin proteins, purified from nerve cell extract, support the anchoring of neurons, and increase neurite sprouting and total neurite outgrowth in culture. These positive effects on neurite regeneration in cell culture suggest that the beta-thymosin proteins have an extracellular function in the central nervous system of Aplysia californica.

Amino Acid Sequence↗

PubMed2005

Subcellular analysis of D-aspartate.

D-Aspartate (D-Asp) is an especially intriguing molecule found within neurons of the central nervous system of animals ranging from mollusks to vertebrates. It has a large variety of roles ascribed to it, including an involvement in cell-to-cell signaling. To determine the D-Asp content in cells and in subcellular domains, a laboratory-assembled capillary electrophoresis system with laser-induced fluorescence (LIF) detection has been used. The system allows chiral separations with sufficient sensitivity and selectivity to measure the D-Asp content in specific subregions of a single neuron, including neuronal processes. The method uses microvial sampling, analyte derivatization with naphthalene-2,3-dicarboxaldehyde, cyclodextrin-mediated micellar electrokinetic capillary chromatography, and sheath flow cell-based LIF detection. Manipulating neuronal processes is difficult as they often disintegrate during the transfer to the sampling vial. We describe a glycerol treatment that stabilizes cell morphology during sample preparation, thereby alleviating the deleterious effects of the high-salt extracellular matrix on the electrophoretic separation. D-Asp percentages in processes from identified neurons from Aplysia californica differ significantly depending on the cell studied. Subcellular analysis reveals more compounds in the cell body than in the processes.

Animals↗

PubMed2005

Confirmation of peak assignments in capillary electrophoresis using immunoprecipitation. Application to D-aspartate measurements in neurons.

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection is a powerful tool for analysis of samples ranging from tissue extracts to single cells. However, accurate peak identification in electropherograms is challenging when complex biological samples are analyzed, as often matching a migration time between an analyte and corresponding standard may be insufficient to confirm the peak's identity. A method which combines single-step immunoprecipitation and CE-LIF analysis for investigation of the chiral amino acids in single cells and small tissue samples is demonstrated. D-Aspartate (D-Asp) has been reported in the central nervous system of the invertebrate neurobiological model Aplysia californica. In order to confirm the identity of D-Asp signal in the complex electropherograms of nerve tissue extracts and individual neurons, anti-D-Asp serum, preincubated with L-Asp conjugate, is added to the sample. This selectively binds the free D-Asp, creating an antibody-antigen complex with a migration time similar to that of antibody alone, but not that of D-Asp. The complete disappearance of the putative D-Asp peak confirms its identity and validates that there are no other detectable analytes co-migrating with D-Asp in the electropherogram.

Animals↗

PubMed2005

Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons.

Self-assembled monolayers (SAMs) of omega-substituted alkanethiols on gold have been explored as well defined in vitro model surfaces for the investigation of neuronal growth and function. When used as cell culture substrates, surfaces with monolayers functionalized with terminal -COOH groups support neuron attachment and growth even without an intermediate protein layer. Addition of a poly-L-lysine layer (PLL) to the -COOH terminated monolayers significantly increases total neurite outgrowth. Mixed monolayers containing -COOH and -CH3 terminal groups in 1:10 and 1:100 ratios poorly support neuron adhesion and preclude neurite extension. A layer of PLL improves the ability of mixed monolayer surfaces to support neuronal growth in culture. The morphology of cultured neurons depends on the chemical composition of SAMs on the support surface. Using glass microelectrode intracellular recording, the properties of cell culture substrates modulate the dynamic properties of action potentials of cultured neurons. These findings provide insight into the cellular responses of excitable cells to the chemical details of a surface and, thus, may help direct the rational design of biologically active materials.

Alkanes↗

PubMed2005

Vitamin E imaging and localization in the neuronal membrane.

Vitamin E (alpha-tocopherol) has been implicated in several cellular processes including signaling, transport, lipid membrane curvature, and several neurodegenerative disorders. Vitamin E imaging has been hindered by the inaccessibility of the molecule to traditional immunohistochemical methods. Using time-of-flight secondary ion mass spectrometry (ToF-SIMS), the distribution of major constituents in the cellular membrane of isolated neurons was investigated. There is a significant increase in the vitamin E signal at the soma-neurite junction compared to the cell as a whole (165 +/- 11% of that found across the cell, p = 0.004, n = 12). The observed membrane distribution suggests an important new role for vitamin E in neuronal function.

Antioxidants↗

PubMed2005

Measuring D-amino acid-containing neuropeptides with capillary electrophoresis.

Neuropeptides are heavily posttranslationally modified (PTM) gene products that are often characterized by a variety of mass spectrometric approaches. Recently, the occurrence of amino acids in the D-form has been documented in several neuropeptides. As this modification has no associated mass shift, this particular PTM is difficult to evaluate using mass spectrometry (MS) alone. Here we demonstrate several approaches using capillary electrophoresis (CE) with absorbance and laser-induced fluorescence (LIF) for the separation of native and derivatized molluscan peptides containing D-amino acids. The combination of peptide derivatization followed by CE/LIF is well suited for single cell measurements because of its ability to characterize the peptides in such small samples. In order to verify this approach, the D-Trp-containing peptide NdWFa (NH2-Asn-D-Trp-Phe-CONH2), present in individual neurons from the marine mollusk Aplysia californica, has been characterized. The mass spectra show that NdWFa and/or NWFa are present in specific neurons; CE/LIF analysis of these cells demonstrates that NdWFa is the dominant form of the peptide.

Amino Acids↗

PubMed2005

Imaging mass spectrometry: fundamentals and applications to drug discovery.

Imaging mass spectrometry (IMS) encompasses a variety of techniques that enable the chemical imaging of analytes, which range in size from atoms and small molecules to intact proteins, directly from biological tissues. IMS is transforming specific areas in biological research with its unique combination of chemical and spatial information. Innovations in instrumentation and imaging protocols will make this approach invaluable at many stages of the drug discovery process, including pharmacological target screening and evaluating the distribution of drug and drug metabolites in cells and tissues. The fundamentals and unique methodology of IMS are discussed, along with exciting new applications to drug discovery science.

Drug Design↗

PubMed2005

The detection of nitrated tyrosine in neuropeptides: a MALDI matrix-dependent response.

Neuropeptides are a diverse class of signaling molecules that typically have one or more posttranslational modifications. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an effective tool for identification and characterization of neuropeptides from samples as small as individual neurons. However, the detection of one particular posttranslational modification-nitrotyrosine-has been problematic because of the lability of the nitro group of nitrotyrosine under MALDI-MS conditions. The detection of nitrated tyrosine in peptide standards was dependent on the MALDI matrix used for the analysis. Specifically, sinapinic acid was the optimum matrix tested to observe this modification while it was not consistently detected with matrices such as 2,5-dihydroxybenzoic acid. Using the optimized procedures, several identified nitric-oxide-synthase positive neurons from Lymnaea stagnalis were tested to determine if the neuropeptides present were nitrated. In all cases, the nitrated form of the neuropeptide was not observed. The dependence on the sample-preparation procedures of observing this particular chemical modification demonstrates the need for careful selection of sample-preparation methods with MALDI or the use of other ionization methods.

Amino Acid Sequence↗

PubMed2005

monitoring activity-dependent peptide release from the CNS using single-bead solid-phase extraction and MALDI TOF MS detection.

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE) beads were used to collect peptides from specific locations of well-characterized neurosecretory structures and even individual neuronal processes for off-line MALDI MS analyses. Peptide binding parameters of single SPE beads, including limits of collection, detection, and saturation capacity, were tested with 14C-labeled cytochrome c as well as with mixtures of multiple neuropeptides (bradykinin, Aplysia acidic peptide 1-20, and insulin). MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysia bag cell neurons. With cultured cells, precise placement of SPE beads allowed peptide collection from distinct neurites with spatial localization on the order of 200 microm, and SPE beads could be replaced within time frames that allowed analyte collection before and after cell stimulation paradigms. Comparison between pre- and poststimulation peptide profiles in both model systems allowed a directed strategy to determine which compounds were released with neuronal activity. Single SPE bead MALDI MS offers a novel approach to investigate peptide signaling that allows the detection and discovery of unknown intercellular signals secreted from a large variety of biological tissues.

Animals↗

PubMed2005

MALDI-MS imaging of features smaller than the size of the laser beam.

The feasibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging of features smaller than the laser beam size has been demonstrated. The method involves the complete ablation of the MALDI matrix coating the sample at each sample position and moving the sample target a distance less than the diameter of the laser beam before repeating the process. In the limit of complete sample ablation, acquiring signal from adjacent positions spaced by distances smaller than the sample probe enhances image resolution as the measured analyte signal only arises from the overlap of the laser beam size and the non-ablated sample surface. Image acquisition of features smaller than the laser beam size has been demonstrated with peptide standards deposited on electron microscopy calibration grids and with neuropeptides originating from single cells. The presented MS imaging technique enables approximately 25 microm imaging spatial resolution using commercial MALDI mass spectrometers having irregular laser beam sizes of several hundred micron diameters. With appropriate sampling, the size of the laser beam is not a strict barrier to the attainable MALDI-MS imaging resolution.

Algorithms↗

PubMed2004

Measurement of nitric oxide by 4,5-diaminofluorescein without interferences.

The fluorescent reagent 4,5-diaminofluorescein (DAF-2) has been widely used for specific and quantitative measurements of nitric oxide (NO) in biological tissues. Recently it was reported that dehydroascorbic acid (DHA) and ascorbic acid (AA) interfere with the measurement of NO using DAF-2. A new method of assaying NO using DAF-2 eliminates these interferences; when frozen on dry ice, the NO in the original solution still diffuses and can react with an adjacent frozen block of DAF-2, but the confounding compounds such as DHA do not. Thus, placing the microliter-volume frozen blocks of solutions containing NO and the solutions of DAF-2 adjacent to each other for 30 min results in the concentration dependent formation of fluorescent product (DAF-2T) from the reaction of NO with DAF-2. The product has been characterized and the method validated using both fluorescence spectroscopy and capillary electrophoresis with laser induced fluorescence detection. With this approach, the presence of DHA and AA does not interfere with NO measurements, and product formation is inhibited in the presence of NO scavengers added to either of the solutions before freezing. The contactless DAF-2 method successfully assays NO in nitric oxide synthase-positive vertebrate and invertebrate tissues. This method allows nondestructive NO detection in biological samples that can subsequently be used for morphological and/or biochemical studies.

Animals↗

PubMed2004

Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning.

The ability to control the orientation, morphology, and electrophysiological characteristics of neurons in culture allows the construction of neural circuits with defined physiological properties. Using microfluidic protein deposition onto chemically modified glass, we achieve the controlled growth of Aplysia neurons on geometrical patterns of poly-L-lysine and collagen IV, surrounded by nonadhesive regions of bovine albumin. We investigate the parameters essential for forming functional neuronal networks, the morphology, biochemistry, and electrophysiology under engineered cell culture conditions. We demonstrate that not only the orientation of neurite extension but also the number of primary neurites originating from the cell soma, their length, and branching pattern depend on the spatial constraints presented by the size and shape of the adhesion region on the patterned substrate. In addition, the physicochemical properties of the support layer influence the electrical activity of the cultured neurons. Substrate-dependent changes in the amplitude and in the dynamic parameters of the action potential cause decreased spike broadening in patterned neurons, which reflects changes in the number or functioning of active membrane ion channels. In contrast to morphology and electrophysiology, the neuropeptide content, as determined by mass spectrometry of individual patterned neurons, is not affected by the growth on patterned surfaces. Our results suggest that the morphological and electrophysiological parameters of neurons can be predictably altered/engineered by modulation of the chemical, physical, and topographical features of culture substrates. We also demonstrate that a full suite of techniques is required for functional characterization of neurons on engineered substrates.

Action Potentials↗

PubMed2004

Characterization of the physicochemical parameters of dense core atrial gland and lucent red hemiduct vesicles in Aplysia californica.

Characterizing femtoliter-volume cellular organelles requires innovative analytical techniques such as mass spectrometry, separations, and NMR. The capabilities of all three are demonstrated for characterizing the physicochemical properties of the electron-dense core atrial gland vesicles from Aplysia californica and comparing them with the same properties of the electron lucent red hemiduct vesicles. Single-vesicle mass spectrometric measurements show that the atrial gland vesicles contain an abundance of peptides while the red hemiduct vesicles contain no detectable peptide signals. Capillary electrophoresis with wavelength-resolved native fluorescence detection is used to characterize larger vesicle samples for tyrosine- and tryptophan-containing peptides. Using NMR spectroscopy, we show that the physiologically active peptides located in the core of the atrial gland vesicles are NMR inactive when the vesicles are intact. Resonances from these peptides appear after vesicle lysis by heating, suggesting that initially they are packed in a crystalline or semicrystalline core so that the NMR resonances are not detectable. In contrast, the red hemiduct vesicles appear to have their contents stored in a completely mobile form due to the fact that no new NMR resonances are detected after heating.

Animals↗