Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20366 (substance P)
 21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Photoreactive analogues of substance P (biotin sulfone-spacer (amino pentanoic or Gly(3))-Arg-Pro-Lys-Pro-(pBzl)Phe-Gln-Phe-Phe-Gly-Leu-Met(O(2))NH(2)) with or without isotope (deuterium) labeling have been synthesized. Deuteriums were present on (d)-biotin or epibiotin sulfone (D(3)), on the Gly(3) spacer linker (D(6)), or on the Gly in position 9 of SP (D(2)). Therefore, peptide analogues could be either unlabeled or tri-, penta-, or hexadeuterated. Results obtained with the use of these peptide analogues show that (d)-biotin sulfone and epibiotin sulfone are not recognized with the same affinity by streptavidin, with (d)-biotin sulfone displaying better affinity for the protein. Photolabeling of the human NK-1 receptor with a 1:1 molar ratio of nondeuterated and deuterated photoreactive substance P (SP) analogues in position 5, followed by combined digestions, purification, and MALDI-TOF mass spectrometry analysis, made the identification of the domain of the receptor covalently linked by the photoreactive SP analogue easier. Indeed, doublets in mass spectra were specific for the covalent complex whereas single peaks could be attributed to contaminating species. This method is particularly suitable when minute amounts of complex have to be analyzed, as in the case of highly hydrophobic G-protein coupled receptors.
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PMID:Isotope and affinity tags in photoreactive substance P analogues to identify the covalent linkage within the NK-1 receptor by MALDI-TOF analysis. 1464 Jul 25

We report here the identification of a tachykinin-related peptide (TRP) in crustaceans. Direct MALDI-TOF MS with brain slices of the crayfish Procambarus clarkii indicated the presence of a peptide having an MS number of around 934. Quadrupole orthogonal acceleration time-of-flight (Q-TOF) MS/MS analysis implied the sequence to be APSGFLGMRamide, identical to that of CabTRP Ia, isolated previously from the crab Cancer borealis, and Pev-tachykinin, from the shrimp Penaeus vannamei. The peptide has been shown to be myoactive in the cockroach hindgut, but the structure of its precursor protein had not been elucidated. A cDNA encoding crayfish preproTRP was cloned, revealing that the 225-residue protein contains seven identical copies of the peptide APSGFLGMRamide. This is unique because TRPs identified in other invertebrates were known to exist in several related forms in each species. The conserved structure of TRP in crustaceans was confirmed by cloning preproTRP from the spiny lobster Panulirus interruptus. RT-PCR and Northern blotting analyses suggested that the crayfish preproTRP mRNA is expressed throughout the nervous system, and in situ hybridization studies of the brain revealed that the transcript predominantly localizes to cell clusters 11 (dorsal lateral cells) and 9 (ventral lateral cells).
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PMID:APSGFLGMRamide is a unique tachykinin-related peptide in crustaceans. 1506 80

Interest in the analysis of low abundance neuropeptides particularly using matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) is increasing because these neuropeptides are essential to the mechanism of transportation and the metabolism. This article describes an immunoprecipitation procedure that is suitable for MALDI-MS analysis of substance P (SP), a neuropeptide, in rat brain tissues. Substance P was precipitated from brain tissue extracts by immunoprecipitation with antibodies directed against SP, and are analyzed by MALDI-TOF-MS. Mass spectrometric analysis showed a singly charged [M+H]+ ion peak that corresponded to the SP molecular mass and was observed with a detection error of 1.6%. The average mass errors between the observed and theoretical molecular mass were within the 0.11 Da range. Capillary zone electrophoresis analysis was subsequently performed, and the effects of the different separation parameters were examined. Beginning with milligram quantities of brain tissue, picomole quantities of SP could be detected using this method.
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PMID:Analysis of substance P in rat brain by means of immunoaffinity capture and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry. 1520 45

Peptides are the largest class of signalling molecules found in animals. Nevertheless, in most proteomic studies peptides are overlooked since they literally fall through the mazes of the net. In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications. In this paper we describe the analysis of the larval fruit fly central nervous system using two-dimensional capillary liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS/MS. Using the central nervous systems of only 50 larval Drosophila as starting material, we identified 38 peptides in a single analysis, 20 of which were not detected in a previous study that reported on the one-dimensional capillary LC/MS/MS analysis of the same tissue. Among the 38 sequenced peptides, some originate from precursors, such as the tachykinin and the IFamide precursor that were entirely missed in the first study. This clearly demonstrates that the two-dimensional capillary LC approach enhances the coverage of the peptidomic analysis.
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PMID:Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole time-of-flight mass spectrometry. 1570 25

Tachykinins and tachykinin-related peptides (TKRPs) play major roles in signaling in the nervous system and intestine of both invertebrates and vertebrates. Here we have identified cDNAs encoding precursors of multiple TKRPs from the cockroaches Leucophaea maderae and Periplaneta americana. All nine LemTKRPs that had been chemically isolated in earlier experiments could be identified on the precursor of L. maderae. Four previously unidentified LemTKRPs were found in addition on the precursor. The P. americana cDNA displayed an open reading frame very similar to that of L. maderae with 13 different TKRPs. MALDI-TOF mass spectra from tissues of both species confirms the presence of all the TKRPs encoded on the precursor plus two additional peptides that are cleavage products of the N-terminally extended TKRPs. A tissue-specific distribution of TKRPs was observed in earlier experiments at isolation from brain and midgut of L. maderae. Our data do not suggest a differential gene expression but a different efficacy in processing of LemTKRP-2 and Lem/PeaTKRP-3 in the brain and intestine, respectively. This results in a gut-specific accumulation of these extended peptides, whereas in the brain their cleavage products, LemTKRP-1 and LemTKRP-3(11-19), are most abundant. Mass spectrometric analysis demonstrated the occurrence of the different TKRPs in single glomeruli of the tritocerebrum and in cells of the optical lobe.
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PMID:Tachykinin-related peptide precursors in two cockroach species. 1597 42

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.
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PMID:Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometry. 1618 23

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.
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PMID:Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS. 1621 43

The neuropeptide profiles and diversity of the brain and retrocerebral organs (corpora cardiaca-corpora allata; CC-CA) of adult workers of the honey bee Apis mellifera carnica (dark European strain) were investigated using a combination of HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with post-source decay (PSD) and collision-induced dissociation (CID) fragmentation. Using evidence from genomic sources, including BLAST searches of the honey bee genome, comparisons with other species and de novo sequencing by PSD and CID fragmentation, a total of 13 mass ions could be assigned to peptides predicted from the A. mellifera genomic database. Peptides positively identified were A. mellifera tachykinin-related peptides 3 and 4 (APMGFQGMRa; APMGFYGTRa) and leucomyosuppressin (pEDVDHVFLRFa). Peptides tentatively identified were A. mellifera tachykinin-related peptides 2 and 5 (ALMGFQGVRa; ARMGFHGMRa), A. mellifera allatostatins 2, 3 and 4 (GRDYSFGLa; RQYSFGLa; GRQPYSFGLa), A1-SIFamide (AYRKPPFNGSIFa), Q1-leucomyosuppressin (QDVDHVFLRFa) and A. mellifera pyrokinins PK 1, PK 2 and Q1-PK 2 (TSQDITSGMWFGPRLa; pEITQFTPRLa; QITQFTPRLa). Allatostatins, tachykinin-related peptides and A1-SIFamide were not detected in CC-CA extract, which appears to contain predominantly leucomyosuppressin, Q1-leucomyosuppressin, PK 1, PK 2, Q1-PK 2 and some unidentified masses. No ion signal was detected that would correspond to the hypertrehalosaemic peptide (=Manse-AKH), which has been isolated from the Italian race of the honey bee (A. mellifera ligustica), but not from A. mellifera carnica.
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PMID:Analysis of peptides in the brain and corpora cardiaca-corpora allata of the honey bee, Apis mellifera using MALDI-TOF mass spectrometry. 1630 91

Recently, phosphoglucose isomerase with a lysyl aminopeptidase (PGI-LysAP) activity was identified in Vibrio vulnificus. In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGI-LysAP of V. vulnificus using three approaches: (i) a quantitative fluorescent ninhydrin assay for free lysine, (ii) matrix-assisted laser desorption ionization-two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and (iii) Tricine gel electrophoresis. PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-(des-Arg9)-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment. We detected the proteolytic release of free L-lysine from peptide digests using a rapid, simple, sensitive, and quantitative fluorescent ninhydrin assay, and results were confirmed by MALDI-TOF-TOF. The use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies. The visualization of peptide hydrolysis was accomplished by Tricine gel electrophoresis. Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability. By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways.
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PMID:Specificity of a Vibrio vulnificus aminopeptidase toward kinins and other peptidyl substrates. 1651 35

Capillary liquid chromatography (CLC) coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS were explored for identification and quantification of neuropeptides in microwave-fixed rat brain tissue. Sample was separated by gradient elution on 50-mum-inner diameter reversed-phase columns at 180 nL/min. Effluent was mixed with matrix solution and transferred to a MALDI target plate by pulsed electric field deposition, yielding sample spots with 200-300-mum diameter. Mass detection limits as low as 2 amol, corresponding to 1 pM concentration, were achieved for neuropeptides. CLC-MALDI-TOF-MS analysis of microwave-fixed rat striatum tissue yielded detection of over 400 distinctive peaks. CLC-MALDI-TOF/TOF-MS allowed identification of 10 peptides including 3 novel peptides. Quantification was evaluated using substance P as analyte and 15N3-labeled substance P as an internal standard. Quantification of substance P revealed approximately 6.8-fold higher levels than previously reported in the rat striatum. This increase is attributed to use of microwave fixation, which prevented degradation of the peptide, aggressive extraction procedures, and accounting for oxidation of substance P in the analysis. These results demonstrate that CLC-MALDI-TOF-MS is a versatile tool for neuropeptide analysis in brain tissue by allowing for detection, identification, and quantification.
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PMID:Identification and quantification of neuropeptides in brain tissue by capillary liquid chromatography coupled off-line to MALDI-TOF and MALDI-TOF/TOF-MS. 1680 41


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