Optimization of immobilized enzyme hydrolysis combined with high-performance liquid chromatography/thermospray mass spectrometry for the determination of neuropeptides

Peptidases, including chymotrypsin, thermolysin, trypsin, V8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7-8 and 40-50 degrees C. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic alpha-, beta-, and gamma-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides' identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current 10-40 times higher than that of the [M + 2H]2+ ion for unhydrolyzed beta-endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol.     

Osmotic lysis of corpora cardiaca using distilled water reveals the presence of partially processed peptides and peptide fragments

Abstract. A simple, single-step aqueous extraction method has been developed to study the neuropeptide content of small neuroendocrine organs. Perifusion of these tissues with deionized water causes osmotic bursting of the cells and release of their content into the surrounding fluid. The neuropeptides are immediately retained from the perifusion fluid using disposable C18 cartridges. After one separation step and mass spectrometry, it was possible to identify a large number of known neuropeptides from the corpora cardiaca of Locusta migratoria (L). Also present in the extract were a number of neuropeptide fragments and two incompletely processed peptides. Using this method, a 959Da peptide present in the corpora cardiaca was sequenced de novo . The full sequence, deduced using Collision Induced Dissociation Tandem Mass Spectrometry (CID MS/MS), is Ser-Pro-Leu-Asp-Ala-His-His-Leu-Ala. This nonapeptide is predicted from the gene encoding the ion transport peptide precursor and from the gene encoding the ion transport-like peptide precursor. In both cases, this nonapeptide, which was named ion transport peptide-copeptide, is flanked by the signal sequence at the N -terminus and a dibasic cleavage site (Lys-Arg) at the C -terminus. This structural feature is common to many physiologically important locust preproneuropeptides and indicates that this copeptide might have a physiological function, but this is not yet known.     

Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules-neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.     

Immunohistochemical identification of calcitonin gene-related peptide and substance P in nerves of the bovine parathyroid gland

Summary Although peptide neurotransmitters have been shown to modulate hormone secretion in many glands, there are very few studies of neurotransmitters in the parathyroid gland. Bovine parathyroid glands were collected at a local abattoir, fixed with paraformaldehyde, sectioned using a cryostat, and stained by indirect immunohistochemistry for calcitonin gene-related peptide and substance P. We were able to positively identify both neuropeptides. Nerve fibres containing calcitonin gene-related peptide and substance P were identified in contact with the tunica media of arteries and arterioles and dispersed throughout the stroma of the gland. While many of the fibres encircled parenchymal lobules, no intimate contact with the peripheral chief cells was observed. All immunoreactive fibres were found to contain both neuropeptides. Since calcitonin gene-related peptide and substance P are vasodilators, they may increase blood flow within the gland. In addition, the neuropeptides may diffuse from perilobular nerve fibres into the parenchyma, thereby modulating secretion of parathyroid hormone.     

Sample Limited Characterization of a Novel Disulfide-Rich Venom Peptide Toxin from Terebrid Marine Snail Terebra variegata

Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.     

Amyloid-Like Fibril Formation by Tachykinin Neuropeptides and Its Relevance to Amyloid β-Protein Aggregation and Toxicity

Protein aggregation and amyloid formation are associated with both pathological conditions in humans such as Alzheimer's disease and native functions such as peptide hormone storage in the pituitary secretory granules in mammals. Here, we studied amyloid fibrils formation by three neuropeptides namely physalaemin, kassinin and substance P of tachykinin family using biophysical techniques including circular dichroism, thioflavin T, congo red binding and microscopy. All these neuropeptides under study have significant sequence similarity with Aβ(25-35) that is known to form neurotoxic amyloids. We found that all these peptides formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells. However, the extent of amyloid formation, structural transition, and morphology were different depending on the primary sequences of peptide. When Aβ(25-35) and Aβ40 were incubated with each of these neuropeptides in 1:1 ratio, a drastic increase in amyloid growths were observed compared to that of individual peptides suggesting that co-aggregation of Aβ and these neuropeptides. The electron micrographs of these co-aggregates were dissimilar when compared with individual peptide fibrils further supporting the possible incorporation of these neuropeptides in Aβ amyloid fibrils. Further, the fibrils of these neuropeptides can seed the fibrils formation of Aβ40 and reduced the toxicity of preformed Aβ fibrils. The present study of amyloid formation by tachykinin neuropeptides is not only providing an understanding of the mechanism of amyloid fibril formation in general, but also offering plausible explanation that why these neuropeptide might reduce the cytotoxicity associated with Alzheimer's disease related amyloids.     

Neuropeptides of the cotton fleahopper, Pseudatomoscelis seriatus (Reuter)

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter), is an economically important pest of cotton, and increasing concerns over resistance, detrimental effects on beneficial insects and safety issues associated with traditional insecticide applications have led to an interest in research on novel, alternative strategies for control. One such approach requires a more basic understanding of the neurohormonal system that regulates important physiological properties of the fleahopper; e.g. the expression of specific messenger molecules such as neuropeptides. Therefore we performed a peptidomic study of neural tissues from the fleahopper which led to the first identification of the sequences of native peptide hormones. These peptide hormones include the following neuropeptides: corazonin, short neuropeptide F (sNPF), myosuppressin, CAPA-pyrokinin and CAPA-PVK peptides. The CAPA-pyrokinin, sNPF, and CAPA-PVK peptides represent novel sequences. A comparison of fleahopper neuropeptides with those of related heteropteran species indicates that they are quite different. The sNPF of P. seriatus shows, among others, a novel substitution of Leu with Phe within the C-terminal region; a modification that sets it apart from the known sNPFs of not only other Heteroptera but of other arthropod species as well. The identity of the neuropeptides native to the fleahopper can aid in the potential development of biostable, bioavailable mimetic agonists and antagonists capable of disrupting the physiological functions that these neuropeptides regulate.     

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography-tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.     

The effects of centrally administered neuropeptides on the development of gastric lesions in the rat

Centrally administered neuropeptides were investigated for their effects on the development of gastric lesions in rats. Thyrotropin releasing hormone (TRH), vasoactive intestinal peptide (VIP) and gonadotropin releasing hormone (LHRH) produced gastric lesions acutely, with TRH demonstrating the most pronounced effect in terms of incidence and severity. Ten-fold higher doses of the same peptides administered intravenously produced none or very few gastric lesions. Moreover, pretreatment with atropine partially inhibited their production. Corticotropin releasing factor (CRF) exhibited only mild ulcerogenic effects, and the gastric lesions induced with this peptide developed more slowly than with TRH, VIP and LHRH. Although ulcerogenic in their own right, none of these four neuropeptides significantly potentiated the potent ulcerogenic effects of cold-restraint stress. Since other neuropeptides, including somatostatin, human pancreatic growth hormone releasing factor (hpGRF), substance P, bombesin, and neurotensin, had no demonstrable effects on gastric mucosa, we can conclude that the lesions were not a general effect of intracisternal administration of neuropeptides. The results suggest that within the central nervous system, there are several neuropeptides that play a significant role in the development of gastric lesions via, at least in part, vagal-dependent mechanisms.     

Selective localization of vasoactive intestinal peptide and substance P in human eosinophils

Extracts of purified human eosinophils had a mean concentration of 72 fmol of immunoreactive vasoactive intestinal peptide and 21 fmol of substance P per 10(7) eosinophils, that were significantly higher than the content of immunoreactivity of the same neuropeptides in neutrophils, mononuclear leukocytes, and platelets. In contrast, the lower concentrations of calcitonin gene-related peptide and somatostatin were similar in extracts of all leukocytes. Chromatography of the peptides from eosinophils confirmed their identity with vasoactive intestinal peptide and substance P from neuroendocrine sources. Stores of some neuropeptides may endow eosinophils with unique roles in host defense and hypersensitivity reactions.     

Segmental distribution of colonic neuropeptides in Hirschsprung's disease.

Despite continued research, the pathophysiologic mechanism responsible for functional obstruction in the aganglionic segment of bowel in Hirschsprung's disease remains controversial. Narrowing of the affected segment is thought by many investigators to be the result of loss of intrinsic inhibitory innervation. For this hypothesis to be consistent, inhibitory neuropeptides should be present in the dilating, transitional segment of bowel. In order to quantitate reported changes in peptidergic nerve staining in Hirschsprung's disease, we measured concentrations of five neuropeptides (vasoactive intestinal peptide, peptide histidine-methionine, met5-enkephalin, substance P and bombesin-like immunoreactivity) by radioimmunoassay in the affected segments of bowel from six patients with Hirschsprung's disease. Tissue extracts were prepared using gut obtained at surgery from the: (1) constricted, aganglionic segment, (2) dilating, aganglionic transitional segment and (3) dilated, proximal ganglionic segment. Concentrations of vasoactive intestinal peptide, peptide histidine-methionine, substance P and met5-enkephalin were significantly reduced in both the muscularis externa and the mucosal-submucosal layers from the constricted aganglionic segment. By contrast, concentrations of the candidate inhibitory neuropeptides, vasoactive intestinal peptide and peptide histidine-methionine, were minimally reduced in the dilating, aganglionic transitional segment. These results are consistent with the hypothesis that constriction of the aganglionic segment is due to loss of intrinsic inhibitory innervation. Concentrations of bombesin-like immunoreactivity were similar in the three segments of human gut, suggesting the presence of this immunoreactive neuropeptide in extrinsic nerve fibers.     

Identification of a platyhelminth neuropeptide receptor

We report the characterisation of the first neuropeptide receptor from the phylum Platyhelminthes, an early-diverging phylum which includes a number of important human and veterinary parasites. The G protein-coupled receptor (GPCR) was identified from the model flatworm Girardia tigrina (Tricladida: Dugesiidae) based on the presence of motifs widely conserved amongst GPCRs. In two different assays utilising heterologous expression in Chinese hamster ovary cells, the Girardia GPCR was most potently activated by neuropeptides from the FMRFamide-like peptide class. The most potent platyhelminth neuropeptide in both assays was GYIRFamide, a FMRFamide-like peptide known to be present in G. tigrina. There was no activation by neuropeptide Fs, another class of flatworm neuropeptides. Also active were FMRFamide-like peptides derived from other phyla but not known to be present in any platyhelminth. Most potent among these were nematode neuropeptides encoded by the Caenorhabditis elegans flp-1 gene which share a PNFLRFamide carboxy terminal motif. The ability of nematode peptides to stimulate a platyhelminth receptor demonstrates a degree of structural conservation between FMRFamide-like peptide receptors from these two distinct, distant phyla which contain parasitic worms.     

Localization of immunoreactive neuropeptides in the kidney of the bullfrog,Rana catesbeiana,by immunofluorescence

Indirect double immunofluorescence labelling for demonstrating nine neuropeptides in the kidney of the bullfrog, Rana catesbeiana, revealed for the first time the occurrence, distribution, and coexistence of certain neuropeptides in the kidney of the submammalian vertebrates. Substance P, neuropeptide Y, and calcitonin generelated peptide were localized in nerve fibers distributed along the afferent arterioles connected with the glomeruli, and along the capillary network between uriniferous tubules. Neuropeptide Y and calcitonin gene-related peptide immunoreactive fibers were more numerous than substance P immunoreactive fibers. In these two regions, about one half of the neuropeptide Y or calcitonin in gene-related peptide fibers contained substance P. No immunoreactivity of vasoactive intestinal polypeptide, somatostatin, FMRFamide, or leucine- and methionine-enkephalins was detected in the bullfrog kidney.     

Imaging mass spectrometry of neuropeptides in decapod crustacean neuronal tissues

Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.     

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.     

Age-dependent levels of plasma neuropeptides in normal children

Several neuropeptides are secreted in high amounts in pediatric tumors such as neuroblastoma and have been used as markers of residual or recurrent disease. Plasma levels of neuropeptides might be expected to change during development, but have not been determined in normal children. We have obtained fresh plasma from cord blood of six full-term infants and from peripheral blood in 41 healthy children, ages 1 month to 21 years. Levels of six neuropeptides, vasoactive intestinal peptide (VIP), somatostatin, gastrin releasing peptide (GRP), substance P, pancreastatin and neuropeptide Y (NPY) were measured by radioimmunoassay along with insulin-like growth factor-1 (IGF-1) whose plasma levels are known to vary during development. A child with neuroblastoma was treated with the somatostatin analogue, octreotide, and the effect on plasma neuropeptides quantified. Octreotide doses of 2-3 microg/kg daily resulted in a 40-60% decrease in plasma levels of IGF-1, pancreastatin and GRP. These results are the first publication of plasma neuropeptide levels in normal children.     

Neuropeptides as Targets for the Development of Anticonvulsant Drugs

Epilepsy is a common neurological disorder characterized by recurrent seizures. These seizures are due to abnormal excessive and synchronous neuronal activity in the brain caused by a disruption of the delicate balance between excitation and inhibition. Neuropeptides can contribute to such misbalance by modulating the effect of classical excitatory and inhibitory neurotransmitters. In this review, we discuss 21 different neuropeptides that have been linked to seizure disorders. These neuropeptides show an aberrant expression and/or release in animal seizure models and/or epilepsy patients. Many of these endogenous peptides, like adrenocorticotropic hormone, angiotensin, cholecystokinin, cortistatin, dynorphin, galanin, ghrelin, neuropeptide Y, neurotensin, somatostatin, and thyrotropin-releasing hormone, are able to suppress seizures in the brain. Other neuropeptides, such as arginine-vasopressine peptide, corticotropin-releasing hormone, enkephalin, β-endorphin, pituitary adenylate cyclase-activating polypeptide, and tachykinins have proconvulsive properties. For oxytocin and melanin-concentrating hormone both pro- and anticonvulsive effects have been reported, and this seems to be dose or time dependent. All these neuropeptides and their receptors are interesting targets for the development of new antiepileptic drugs. Other neuropeptides such as nesfatin-1 and vasoactive intestinal peptide have been less studied in this field; however, as nesfatin-1 levels change over the course of epilepsy, this can be considered as an interesting marker to diagnose patients who have suffered a recent epileptic seizure.     

Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides

Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well-characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re-evaluated. We investigate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides in Callinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms to increase almost two-fold, from 18 to 36. Product ion-triggered electron-transfer/higher-energy collision dissociation enabled the site-specific detection of 55 intact N- and O-linked glycoforms, while the faster stepped collision energy higher-energy collisional dissociation resulted in detection of 25. Additionally, applying this workflow to five neuronal tissues enabled the characterization of 36 more glycoforms of known neuropeptides and 11 more glycoforms of nine putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro- and micro-heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides.