RESUMEN
The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.
Asunto(s)
Braquiuros/metabolismo , FMRFamida/metabolismo , Pericardio/metabolismo , Secuencia de Aminoácidos , Animales , FMRFamida/química , Análisis de Fourier , Inmunoprecipitación , Pericardio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
A combined approach using mass spectrometry, a novel neuron affinity capture technique, and Drosophila melanogaster genetic manipulation has been developed to characterize the expression and localization of neuropeptides in the adult D. melanogaster brain. In extract from the whole adult brain, 42 neuropeptides from 18 peptide families were sequenced. Neuropeptide profiling also was performed on targeted populations of cells which were enriched with immunoaffinity purification using a genetically expressed marker.
Asunto(s)
Encéfalo/metabolismo , Drosophila melanogaster/metabolismo , Neuropéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/ultraestructura , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/ultraestructura , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Braquiuros/química , Espectrometría de Masas/métodos , Tejido Nervioso/química , Neuropéptidos/química , Isoformas de Proteínas/química , Secuencia de Aminoácidos , Animales , Braquiuros/anatomía & histología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Over a quarter of a century ago, Mykles described the presence of putative endocrine cells in the midgut epithelium of the crab Cancer magister (Mykles, 1979). In the years that have followed, these cells have been largely ignored and nothing is known about their hormone content or the functions they play in this species. Here, we used a combination of immunohistochemistry and mass spectrometric techniques to investigate these questions. Using immunohistochemistry, we identified both SIFamide- and tachykinin-related peptide (TRP)-like immunopositive cells in the midgut epithelium of C. magister, as well as in that of Cancer borealis and Cancer productus. In each species, the SIFamide-like labeling was restricted to the anterior portion of the midgut, including the paired anterior midgut caeca, whereas the TRP-like immunoreactivity predominated in the posterior midgut and the posterior midgut caecum. Regardless of location, label or species, the morphology of the immunopositive cells matched that of the putative endocrine cells characterized ultrastructurally by Mykles (Mykles, 1979). Matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry identified the peptides responsible for the immunoreactivities as GYRKPPFNGSIFamide (Gly1-SIFamide) and APSGFLGMRamide [Cancer borealis tachykinin-related peptide Ia (CabTRP Ia)], respectively, both of which are known neuropeptides of Cancer species. Although the function of these midgut-derived peptides remains unknown, we found that both Gly1-SIFamide and CabTRP Ia were released when the midgut was exposed to high-potassium saline. In addition, CabTRP Ia was detectable in the hemolymph of crabs that had been held without food for several days, but not in that of fed animals, paralleling results that were attributed to TRP release from midgut endocrine cells in insects. Thus, one function that midgut-derived CabTRP Ia may play in Cancer species is paracrine/hormonal control of feeding-related behavior, as has been postulated for TRPs released from homologous cells in insects.
Asunto(s)
Braquiuros/química , Células Enteroendocrinas/química , Neuropéptidos/genética , Oligopéptidos/genética , Secuencia de Aminoácidos , Animales , Células Enteroendocrinas/ultraestructura , Inmunohistoquímica , Microscopía Fluorescente , Datos de Secuencia Molecular , Neuropéptidos/análisis , Oligopéptidos/análisis , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Methyl esterification of a peptide converts carboxylic acids, such as those present on the side chains of aspartic (D) and glutamic acid (E) as well as the free carboxyl terminus, to their corresponding methyl esters. This method has been applied to peptide and protein quantitation, de novo sequencing, and reduction of nonspecific binding in immobilized metal affinity chromatography for enrichment of phosphorylated peptides. In this study, we investigate the application of this derivatization reaction to the identification and characterization of the orcokinin neuropeptide family by screening and localizing the acidic side chains in peptides. The methyl esterification reaction drastically improves the fragmentation efficiency of modified orcokinins due to blockage of the aspartate selective cleavage pathway of the native orcokinin peptides. With the improved sustained off-resonance irradiation-collisional-induced dissociation spectra, the number and the locations of D and E residues are easily deduced. In addition, a side reaction that occurs at the carboxamide group of asparagine (N) is studied. The deamidation followed by subsequent methyl esterification reaction mechanism is proposed based on the study of an isotope-labeled standard N*FDEIDR. Reaction kinetics is studied by elevating the temperature from room temperature to 37 degrees C. The deamidation-methyl esterification products are greatly enhanced with elevated reaction temperature. Furthermore, we also explore the utility of this side reaction for rapid screening and characterization of C-terminally amidated neuropeptides. This derivatization reaction is applied to both in situ direct tissue neuropeptide analysis and the analysis of HPLC fractions from the separation of complex neuronal tissue extracts. Overall, this study reports a simple and effective method for profiling and localizing acidic amino acid residues (D/E), amide-containing residues (N/Q), and the C-terminal amide group in a peptide.
Asunto(s)
Metanol/química , Neuropéptidos/análisis , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Animales , Esterificación , Datos de Secuencia Molecular , Neuropéptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Pyrokinin (PK) peptides localize to the central and peripheral nervous systems of arthropods, but their actions in the CNS have yet to be studied in any species. Here, we identify PK peptide family members in the crab Cancer borealis and characterize their actions on the gastric mill (chewing) and pyloric (filtering) motor circuits in the stomatogastric ganglion (STG). We identified PK-like immunolabeling in the STG neuropil, in projection neuron inputs to this ganglion, and in the neuroendocrine pericardial organs. By combining MALDI mass spectrometry (MS) and ESI tandem MS techniques, we identified the amino acid sequences of two C. borealis pyrokinins (CabPK-I, CabPK-II). Both CabPKs contain the PK family-specific carboxy-terminal amino acid sequence (FXPRLamide). PK superfusion to the isolated STG had little influence on the pyloric rhythm but excited many gastric mill neurons and consistently activated the gastric mill rhythm. Both CabPKs had comparable actions in the STG and these actions were equivalent to those of Pevpyrokinin (shrimp) and Leucopyrokinin (cockroach). The PK-elicited gastric mill rhythm usually occurred without activation of the projection neuron MCN1. MCN1, which does not contain CabPKs, effectively drives the gastric mill rhythm and at such times is also a gastric mill central pattern generator (CPG) neuron. Because the PK-elicited gastric mill rhythm is independent of MCN1, the underlying core CPG of this rhythm is different from the one responsible for the MCN1-elicited rhythm. Thus neuromodulation, which commonly alters motor circuit output without changing the core CPG, can also change the composition of this core circuit.
Asunto(s)
Braquiuros/metabolismo , Ganglios de Invertebrados/metabolismo , Masticación/fisiología , Neuronas Motoras/metabolismo , Sistema Nervioso/metabolismo , Neuropéptidos/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/fisiología , Braquiuros/citología , Sistema Digestivo/inervación , Conducta Alimentaria/efectos de los fármacos , Conducta Alimentaria/fisiología , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/efectos de los fármacos , Inmunohistoquímica , Masculino , Neuronas Motoras/efectos de los fármacos , Red Nerviosa/citología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/efectos de los fármacos , Neuropéptidos/aislamiento & purificación , Neuropéptidos/farmacología , PeriodicidadRESUMEN
The stomatogastric ganglion (STG) and the cardiac ganglion (CG) of decapod crustaceans are modulated by neuroactive substances released locally and by circulating hormones released from neuroendocrine structures including the pericardial organs (POs). Using nanoscale liquid chromatography electrospray ionization quadrupole-time-of-flight tandem mass spectrometry and direct tissue matrix-assisted laser desorption/ionization Fourier transform mass spectrometry we have identified and sequenced a novel neuropeptide, GAHKNYLRFamide (previously misassigned as KHKNYLRFamide in a study that did not employ peptide derivatization), from the POs and/or the stomatogastric nervous system (STNS) of the crabs, Cancer borealis, Cancer productus and Cancer magister. In C. borealis, exogenous application of GAHKNYLRFamide increased the burst frequency and number of spikes per burst of the isolated CG and re-initiated bursting activity in non-bursting ganglia, effects also elicited by the FMRFamide-like peptides (FLPs) SDRNFLRFamide and TNRNFLRFamide. In the intact STNS (which contains the STG), exogenous application of GAHKNYLRFamide increased the frequency of the pyloric rhythm and activated the gastric mill rhythm, effects also similar to those elicited by SDRNFLRFamide and TNRNFLRFamide. FLP-like immunoreactivity in the POs and the STNS was abolished by pre-adsorption with the synthetic GAHKNYLRFamide. Different members of the FLP family exhibited differential degradation in the presence of extracellular peptidases. Taken collectively, the amino acid sequence of GAHKNYLRFamide, the blocking of FLP-like immunostaining, and its physiological effects on the CG and STNS suggest that this peptide is a novel member of the FLP superfamily.