Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold.

Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg(50) residue to generate a smaller 49-residue mature peptide. The Arg(50) cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly alpha-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.

Two toxins from Conus striatus that individually induce tetanic paralysis.

We describe structural properties and biological activities of two related O-glycosylated peptide toxins isolated from injected (milked) venom of Conus striatus, a piscivorous snail that captures prey by injecting a venom that induces a violent, spastic paralysis. One 30 amino acid toxin is identified as kappaA-SIVA (termed s4a here), and another 37 amino acid toxin, s4b, corresponds to a putative peptide encoded by a previously reported cDNA. We confirm the amino acid sequences and carry out structural analyses of both mature toxins using multiple mass spectrometric techniques. These include electrospray ionization ion-trap mass spectrometry and nanoelectrospray techniques for small volume samples, as well as matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis as a complementary method to assist in the determination of posttranslational modifications, including O-linked glycosylation. Physiological experiments indicate that both s4a and s4b induce intense repetitive firing of the frog neuromuscular junction, leading to a tetanic contracture in muscle fiber. These effects apparently involve modification of voltage-gated sodium channels in motor axons. Notably, application of either s4a or s4b alone mimics the biological effects of the whole injected venom on fish prey.

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.

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.

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.

The first gamma-carboxyglutamate-containing neuropeptide.

A key factor in the characterization of peptide transmitters used in neuronal signaling is the correct elucidation of post-translational modifications, especially as they are often required to confer biological activity. A rare carboxylation modification is described on the D-peptide from the insulin prohormone in the sea slug, Aplysia californica. Using liquid chromatography purification coupled with electrospray ionization and nanoelectrospray ionization-ion trap-mass spectrometry (ESI- and nanoESI-MS), the presence of this D-peptide within Aplysia insulin (AI)-producing neurons is confirmed. Further detailed mass spectrometric analyses demonstrate that the Aplysia insulin D-peptide is carboxylated on the single glutamate residue within the sequence. This gamma-carboxy D-peptide, along with other identified AI-related peptides, is secreted from the central nervous system in response to ionophore stimulation, thus suggesting a signaling role within the nervous system. Although carboxylated peptides have been described previously, the Aplysia gamma-carboxy D-peptide appears to be the first reported carboxylated neuropeptide.

A multilayer poly(dimethylsiloxane) electrospray ionization emitter for sample injection and online mass spectrometric detection.

An ESI emitter made of poly(dimethylsiloxane) interfaces on-chip sample preparation with MS detection. The unique multilayer design allows both the analyte and the spray solutions to reside on the device simultaneously in discrete microfluidic environments that are spatially separated by a polycarbonate track-etched, nanocapillary array membrane (NCAM). In direct spray mode, voltage is applied to the microchannel containing a spray solution delivered via a syringe pump. For injection, the spray potential is lowered and a voltage is applied that forward biases the membrane and permits the analyte to enter the spray channel. Once the injection is complete, the bias potential is switched off, and the spray voltage is increased to generate the ESI of the injected analyte plug. Consecutive injections of a 10 microM bovine insulin solution are reproducible and produce sample plugs with limited band broadening and high quality mass spectra. Peptide signals are observed following transport through the NCAM, even when the peptide is dissolved in solutions containing up to 20% seawater. The multilayer emitter shows great potential for performing multidimensional chemical manipulations on-chip, followed by direct ESI with negligible dead volume for online MS analysis.

Intraspecific variation of venom injected by fish-hunting Conus snails.

Venom peptides from two species of fish-hunting cone snails (Conus striatus and Conus catus) were characterized using microbore liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and electrospray ionization-ion trap-mass spectrometry. Both crude venom isolated from the venom duct and injected venom obtained by milking were studied. Based on analysis of injected venom samples from individual snails, significant intraspecific variation (i.e. between individuals) in the peptide complement is observed. The mixture of peptides in injected venom is simpler than that in the crude duct venom from the same snail, and the composition of crude venom is more consistent from snail to snail. While there is animal-to-animal variation in the peptides present in the injected venom, the composition of any individual's injected venom remains relatively constant over time in captivity. Most of the Conus striatus individuals tested injected predominantly a combination of two neuroexcitatory peptides (s4a and s4b), while a few individuals had unique injected-venom profiles consisting of a combination of peptides, including several previously characterized from the venom duct of this species. Seven novel peptides were also putatively identified based on matches of their empirically derived masses to those predicted by published cDNA sequences. Profiling injected venom of Conus catus individuals using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry demonstrates that intraspecific variation in the mixture of peptides extends to other species of piscivorous cone snails. The results of this study imply that novel regulatory mechanisms exist to select specific venom peptides for injection into prey.

Online microdialysis-dynamic nanoelectrospray ionization-mass spectrometry for monitoring neuropeptide secretion.

Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.

Sequencing and mass profiling highly modified conotoxins using global reduction/alkylation followed by mass spectrometry.

A novel high-throughput method for characterizing heavily modified peptides from cone snail venom is described. Unpurified cone snail duct venom, consisting primarily of multiply disulfide-bonded peptides, is reduced and alkylated using a global procedure in order to simultaneously reduce and derivatize dozens of disulfide-bonded peptides. Samples of Conus victoriae venom are analyzed by online liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-MS) with collisionally induced dissociation (CID). Comparison of the mass profiles of peptides and CID spectra before and after the global reduction and alkylation enables cysteine-containing conopeptides to be ascertained. In this case, over 40 conotoxins are characterized based on only two LC-ESI-MS experiments in terms of mass, number of disulfide-linked cysteine residues (and hence, potential toxin superfamilies), relative hydrophobicity, and other posttranslational modifications. Using this technique, over half of the amino acids (by mass) of several peptides are defined prior to any detailed sequencing studies. Further comparison of the mass data with previously published genetic information allows sequence verification of three novel peptides, termed vc5b, vc6b and vc6c, based on both LC-ESI-MS CID and nanoelectrospray ionization-ion trap-mass spectrometry (nanoESI-MS) experiments. This global method is ideally suited to the use of larger genetic databases in order to efficiently sequence peptides in Conus venoms and is also applicable to analysis of other disulfide-rich classes of peptides such as defensins, chemokines, and snake, spider, or other venoms.

Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry.

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.

Quantitative submonolayer spatial mapping of Arg-Gly-Asp-containing peptide organomercaptan gradients on gold with matrix-assisted laser desorption/ionization mass spectrometry.

Peptides containing the tripeptide sequence Arg-Gly-Asp (RGD) have the ability to bind to members of the integrin superfamily of cell-surface receptors and direct cellular adhesion and haptotaxis. The goal of this work is the development of a rapid and effective method for the quantitative submonolayer spatial composition mapping of surfaces displaying molecular assemblies of RGD-containing organomercaptan peptides on a Au surface using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS). Quantitation of the RGD peptide is achieved by determining the peak intensity of the protonated molecular ion, (M + H)+, relative to the (M + H)+ peak for an internal standard, which is similar chemically but with glutamic acid (E) substituted for aspartic acid (D). Using optimized sample preparation procedures, a bilinear calibration was obtained between the quantitative peak intensity ratio and the mole fraction of the RGD-containing peptide. Quantitative compositions were determined with relative standard deviations of <10%, even in the presence of 10x spot-to-spot variations in the absolute signal intensities, by using this internal standard approach. This MALDI-MS quantitative analysis method was employed to probe variable-width two-component counterpropagating electrochemically generated gradients of the two peptides, prepared by coupling in-plane electrochemical potential gradients with the electrosorption reactions of organothiols to vary the composition laterally. The measured lateral composition profiles match the quasi-linear potential gradient model and yield profiles that overlap to a high degree of fidelity in potential space. Thus, MALDI-MS spatial composition mapping should become a powerful tool for the preparation of designed surfaces facilitating the study of cellular adhesion and motility and cell-cell interactions.

Serotonin catabolism depends upon location of release: characterization of sulfated and gamma-glutamylated serotonin metabolites in Aplysia californica.

Serotonin is a vital neurotransmitter for the functioning of the nervous system in species throughout the animal phyla. Despite its ubiquitous nature, the metabolism of this molecule has yet to be completely elucidated in even the most basic of organisms. Two novel serotonin catabolites, serotonin-O-sulfate and gamma-glu-serotonin-O-sulfate, are chemically characterized using capillary electrophoresis with wavelength-resolved fluorescence detection and electrospray mass spectrometry, and the formation of gamma-glu-serotonin in Aplysia californica is confirmed. These novel compounds appear to be synthesized enzymatically, and known mammalian enzymes exist for all serotonin transformations observed here. The pathway of serotonin inactivation depends upon the type of neuronal tissue subjected to neurotransmitter incubation, with assorted serotonin products observed in distinct locations. Initially demonstrated to be in the metacerebral cell (MCC) soma, the new serotonin metabolite serotonin-O-sulfate may contribute to important functions in the serotonergic system beyond simple serotonin inactivation.