To investigate bioequivalence (BE) testing of an acarbose formulation in healthy Chinese volunteers through the use of recommended and innovative pharmacodynamic (PD) parameters. Following the Food and Drug Administration (FDA) guidance, a randomized, cross-over study of acarbose test (T) and reference (R) (Glucobay®) formulations was performed with a 1-week wash-out period. Preliminary pilot studies showed that the appropriate dose of acarbose was 2 × 50 mg, and the required number of subjects was 40. Serum glucose concentrations after sucrose administration (baseline) and co-administration of sucrose/acarbose on the following day were both determined. Three newly defined PD measures of glucose fluctuation (glucose excursion (GE), GE′ (glucose excursion without the effect of the homeostatic glucose control), and fAUC (degree of fluctuation of serum glucose based on AUC)), the plateau glucose concentration (Css), and time of maximum reduction in glucose concentration (Tmax) were tested in the evaluation. The adequacy of the two parameters recommended by the FDA, ΔCSG,max (maximum reduction in serum glucose concentration) and AUEC(0-4h) (reduction in the AUC(0-4h) of glucose between baseline and acarbose formulation) was also evaluated. The Tmax values were comparable, and the 90% confidence intervals of the geometric test/reference ratios (T/R) for ΔCSG,max, Css, GE, and fAUC were all within 80–125%. The parameter GE′ was slightly outside the limits, and the parameter AUEC(0-4h) could not be computed due to the presence of negative values. In acarbose BE evaluation, while the recommended parameter ΔCSG,max is valuable, the combination of Css and one of the newly defined glucose fluctuation parameters, GE, GE’, and fAUC is preferable than AUEC(0-4h). The acarbose test formulation can be initially considered to be bioequivalent to Glucobay®.
acarbose; bioequivalence (BE); degree of fluctuation of serum glucose based on AUC (fAUC); glucose excursion (GE); pharmacodynamic
IPG-CIEF; MALDI; neuropeptide; monolith; GMA-DVB
Shotgun proteomics commonly utilizes database search like Mascot to identify proteins from tandem MS/MS spectra. False discovery rate (FDR) is often used to assess the confidence of peptide identifications. However, a widely accepted FDR of 1% sacrifices the sensitivity of peptide identification while improving the accuracy. This article details a machine learning approach combining retention time based support vector regressor (RT-SVR) with q value based statistical analysis to improve peptide and protein identifications with high sensitivity and accuracy. The use of confident peptide identifications as training examples and careful feature selection ensures high R values (>0.900) for all models. The application of RT-SVR model on Mascot results (p=0.10) increases the sensitivity of peptide identifications. q value, as a function of deviation between predicted and experimental RTs(Δ RT), is used to assess the significance of peptide identifications. We demonstrate that the peptide and protein identifications increase by up to 89.4% and 83.5%, respectively, for a specified q value of 0.01 when applying the method to proteomic analysis of the natural killer leukemia cell line (NKL). This study establishes an effective methodology and provides a platform for profiling confident proteomes in more relevant species as well as a future investigation of accurate protein quantification.
tandem mass spectrometry; shotgun proteomics; database search; support vector regressor; retention time; q value; peptide identification; NKL cell
Tachykinin-related peptide (TRP) refers to a large and structurally diverse family of neuropeptides found in vertebrate and invertebrate nervous systems. These peptides have various important physiological functions, from regulating stress in mammals to exciting the pyloric (food filtering) rhythm in the stomatogastric nervous system (STNS) of decapod crustaceans. Here, a novel TRP, which we named CalsTRP (Callinectes sapidus TRP), YPSGFLGMRamide (m/z 1026.52), was identified and de novo sequenced using a multifaceted mass spectrometry-based platform in both the central nervous system (CNS) and STNS of C. sapidus. We also found, using isotopic formaldehyde labeling, that CalsTRP in the C. sapidus brain and commissural ganglion (CoG) was up-regulated after food-intake, suggesting that TRPs in the CNS and STNS are involved in regulating feeding in Callinectes. Using imaging mass spectrometry, we determined that the previously identified CabTRP Ia (APSGFLGMRamide) and CalsTRP were co-localized in the C. sapidus brain. Lastly, our electrophysiological studies show that bath-applied CalsTRP and CabTRP Ia each activates the pyloric and gastric mill rhythms in C. sapidus, as shown previously for pyloric rhythm activation by CabTRP Ia in the crab Cancer borealis. In summary, the newly identified CalsTRP joins CabTRP Ia as a TRP family member in the decapod crustacean nervous system, whose actions include regulating feeding behavior.
Neuropeptide; CalsTRP; Callinectes sapidus; tachykinin-related peptide; mass spectrometry; feeding; stomatogastric nervous system
Tachykinin-related peptide (TRP) refers to a large and
diverse family of neuropeptides found in vertebrate and invertebrate
nervous systems. These peptides have various important physiological
functions, from regulating
stress in mammals to exciting the gastric mill (food chewing) and pyloric (food filtering) rhythm
in the stomatogastric nervous system (STNS) of decapod crustaceans.
Here, a novel TRP, which we named CalsTRP (Callinectes
sapidus TRP), YPSGFLGMRamide (m/z 1026.52), was identified and de novo sequenced using a
multifaceted mass spectrometry-based platform in both the central
nervous system (CNS) and STNS of C. sapidus. We also found, using isotopic formaldehyde labeling, that CalsTRP
in the C. sapidus brain and commissural
ganglion (CoG) was up-regulated after food intake, suggesting that
TRPs in the CNS and STNS are involved in regulating feeding in Callinectes. Using imaging mass spectrometry, we
determined that the previously identified CabTRP Ia (APSGFLGMRamide)
and CalsTRP were colocalized in the C. sapidus brain. Lastly, our electrophysiological studies show that bath-applied
CalsTRP and CabTRP Ia each activate the pyloric and gastric mill rhythms
in C. sapidus, as shown previously
for pyloric rhythm activation by CabTRP Ia in the crab Cancer borealis. In summary, the newly identified
CalsTRP joins CabTRP Ia as a TRP family member in the decapod crustacean
nervous system, whose actions include regulating feeding behavior.
Neuropeptide; CalsTRP; Callinectes
sapidus; tachykinin-related peptide; mass
spectrometry; feeding; stomatogastric nervous system
Mass spectrometric imaging (MSI) is a powerful analytical technique that provides two- and three-dimensional spatial maps of multiple compounds in a single experiment. This technique has been routinely applied to protein, peptide, and lipid molecules with much less research reporting small molecule distributions, especially pharmaceutical drugs. This review’s main focus is to provide readers with an up-to-date description of the substrates and compounds that have been analyzed for drug and metabolite composition using MSI technology. Additionally, ionization techniques, sample preparation, and instrumentation developments are discussed.
Drug; Pharmaceuticals; Metabolite; MALDI; SIMS; NIMS; DESI; LAESI; Mass spectrometry; Imaging mass spectrometry (IMS); Mass spectrometric imaging (MSI)
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, one novel orcomyotropin, one novel Ork/Orcomyotropin-related and one 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 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 of the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.
De novo sequencing; Callinectes sapidus; sinus glands; mass spectrometry; neuropeptides; CPRP; Orcokinins; capillary isoelectric focusing; CIEF; ETD; CID
The crustacean stomatogastric ganglion (STG) is modulated by a large number of amines and neuropeptides that are found in descending pathways from anterior ganglia or reach the STG via the hemolymph. Among these are the allatostatin (AST) – B types also known as myoinhibitory peptides (MIPs). We used mass spectrometry to determine the sequences of nine members of the AST-B family of peptides that were found in the stomatogastric nervous system of the crab, Cancer borealis. We raised an antibody against Cancer borealis Allatostatin-B1 (CbAST-B1) (VPNDWAHFRGSWa) and used it to map the distribution of CbAST-B1-like immunoreactivity (-LI) in the stomatogastric nervous system. CbAST-B1-LI was found in neurons and neuropil in the commissural ganglia (CoGs), in somata in the esophageal ganglion (OG), in fibers in the stomatogastric nerve (stn), and in neuropilar processes in the STG. CbAST-B1-LI was blocked by preincubation with 10-6 M CbAST-B1, and partially blocked by lower concentrations. Electrophysiological recordings of the effects of CbAST-B1, CbAST-B2, and CbAST-B3 on the pyloric rhythm of the STG showed that all three peptides inhibited the pyloric rhythm in a state-dependent manner. Specifically, all three peptides at 10-8 M significantly decreased the frequency of the pyloric rhythm when the initial frequency of the pyloric rhythm was below 0.6 Hz. These data suggest important neuromodulatory roles for the CbAST-B family in the stomatogastric nervous system.
neuropeptides; peptide sequencing; immunocytochemistry; pyloric rhythm; matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry (MALDI TOF/TOF MS)
Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis.
capillary isoelectric focusing; CIEF; MALDI-FTMS; mass spectrometry; neuropeptides; orcokinin
Mass spectrometry (MS) – based proteomic approaches have evolved as powerful tools for the discovery of biomarkers. However, the identification of potential protein biomarkers from biofluid samples is challenging because of the limited dynamic range of detection. Currently there is a lack of sensitive and reliable pre-mortem diagnostic test for prion diseases. Here, we describe the use of a combined MS-based approach for biomarker discovery in prion diseases from mouse plasma samples. To overcome the limited dynamic range of detection and sample complexity of plasma samples, we used lectin affinity chromatography and multi-dimensional separations to enrich and isolate glycoproteins at low abundance. Relative quantitation of a panel of proteins was obtained by a combination of isotopic labeling and validated by spectral counting. Overall 708 proteins were identified, 53 of which showed more than 2-fold increase in concentration whereas 58 exhibited more than 2-fold decrease. A few of the potential candidate markers were previously associated with prion or other neurodegenerative diseases.
Prion disease; biomarkers; glycoprotein; mass spectrometry; proteomics; quantitation; multi-dimensional separation
In this work, the utilization of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by TOF/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.
Three dimensional mass spectral imaging (3D MSI) is an exciting field that grants the ability to study a broad mass range of molecular species ranging from small molecules to large proteins by creating lateral and vertical distribution maps of select compounds. Although the general premise behind 3D MSI is simple, factors such as choice of ionization method, sample handling, software considerations and many others must be taken into account for the successful design of a 3D MSI experiment. This review provides a brief overview of ionization methods, sample preparation, software types and technological advancements driving 3D MSI research of a wide range of low- to high-mass analytes. Future perspectives in this field are also provided to conclude that the positive and promises ever-growing applications in the biomedical field with continuous developments of this powerful analytical tool.
Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization has been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that under certain concentrations, detergents can minimize the nonspecific bindings and facilitate the elution of hydrophobic glycoproteins. With the Detergent Assisted Lectin Affinity Chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. 699 proteins were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.
Brain; Detergent; Glycoproteins; Lectin Affinity Chromatography; Membrane Proteins; Mass Spectrometry
Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides.
Hepatitis E is a worldwide public health problem, especially in areas with poor sanitation. This study examines the potential hepatitis E virus (HEV) animal reservoirs and the current status of HEV infection among animals and humans in an endemic area of Xinjiang, China. One thousand five hundred twenty-one serum samples from 12 different animal species and 296 sera from humans were detected for anti-HEV with an in-house enzyme immunoassay, and partial HEV RNA was amplified with a reverse transcription–nested polymerase chain reaction (RT-nPCR). All these distinct animal species, except jerboa and hoptoad, were positive for anti-HEV. However, HEV RNA was only amplified from pigs and a sporadic hepatitis E case in humans. The human HEV strain (CHN-XJ-HE29) shared 100% nucleotide identity with the swine HEV strain (CHN-XJ-SW50), both of which were collected from the same district; this indicates the possibility of HEV transmission from swine to humans in an endemic area.
Herein we describe the development and application of a set of novel N, N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2) tagging reagents with high quantitation efficacy and greatly reduced cost for neuropeptide and protein analysis. DiLeu reagents serve as attractive alternatives for isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency and improved fragmentation efficiency. DiLeu reagent resembles the general structure of a tandem mass tag in that it contains an amine reactive group (triazine ester) targeting the N-terminus and ε-amino group of the lysine side-chain of a peptide, a balance group, and a reporter group. A mass shift of m/z 145.1 is observed for each incorporated label. Intense a1 reporter ions at m/z 115.1, 116.1, 117.1, and 118.1 are observed for all pooled samples upon MS2. All labeling reagents are readily synthesized from commercially available chemicals with greatly reduced cost. Labels 117 and 118 can be synthesized in one step and labels 115 and 116 can be synthesized in two steps. Both DiLeu and iTRAQ reagents show comparable protein sequence coverage (~43%) and quantitation accuracy (<15%) for tryptically digested protein samples. Furthermore, enhanced fragmentation of DiLeu labeling reagents offers greater confidence in protein identification and neuropeptide sequencing from complex neuroendocrine tissue extracts from a marine model organism, Callinectes sapidus.
tandem mass tag; quantitation; isobaric tagging reagents; stable isotope labeling; peptidomics
Feeding behavior is a fundamental aspect of energy homeostasis and is crucial for animal survival. This process is regulated by a multitude of neurotransmitters including neuropeptides within a complex neuroendocrine system. Given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. Here we report comparative neuropeptidomic analysis of brain and major neuroendocrine organ in a crustacean model organism in response to feeding. A multi-faceted approach employing direct tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), stable isotopic labeling of neuropeptide extracts for quantitation, and mass spectrometric imaging (MSI) has been employed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and the pericardial organ (PO) in the crab Cancer borealis. Multiple neuropeptides exhibited changes in abundance after feeding, including RFamides, Cancer borealis tachykinin related peptides (CabTRPs), RYamides, and pyrokinins. By combining quantitative analysis of neuropeptide changes via isotopic labeling of brain extract and MSI mapping of neuropeptides of brain slices, we identified the boundary of olfactory lobe (ON) and median protocerebrum (MPC) area as two potential feeding centers in the crab brain.
Feeding; Neuropeptide; Cancer borealis; Quantitation; MALDI mass spectrometric imaging (MSI); MALDI-TOF/TOF
Feeding behavior is a fundamental aspect of energy homeostasis and is crucial for animal survival. This process is regulated by a multitude of neurotransmitters including neuropeptides within a complex neuroendocrine system. Given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. Here we report comparative neuropeptidomic analysis of brain and a major neuroendocrine organ in a crustacean model organism in response to feeding. A multifaceted approach employing direct tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), stable isotopic labeling of neuropeptide extracts for quantitation, and mass spectrometric imaging (MSI) has been employed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and the pericardial organ (PO) in the crab Cancer borealis. Multiple neuropeptides exhibited changes in abundance after feeding, including RFamides, Cancer borealis tachykinin-related peptides (CabTRPs), RYamides, and pyrokinins. By combining quantitative analysis of neuropeptide changes via isotopic labeling of brain extract and MSI mapping of neuropeptides of brain slices, we identified the boundary of the olfactory lobe (ON) and the median protocerebrum (MPC) area as two potential feeding centers in the crab brain.
Feeding; neuropeptide; Cancer borealis; quantitation; MALDI mass spectrometric imaging (MSI); MALDI-TOF/TOF
The lobster Homarus americanus has long served as an important animal model for electrophysiological and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biological functions. Using nanoLC ESI Q-TOF MS/MS and three types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/extract analysis and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resolution and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide analysis in terms of peptidome coverage, sensitivity, mass spectral resolution and capability for de novo sequencing.
Homarus americanus; MALDI FTICR MS; MALDI TOF/TOF; nanoLC ESI QTOF; MALDI LTQ Orbitrap; pericardial organ; neuropeptide; bioinformatics; mass spectrometric imaging (MSI)
The shrimp Litopenaeus vannamei is arguably the most important aquacultured crustacean, being the subject of a multi-billion dollar industry worldwide. To extend our knowledge of peptidergic control in this species, we conducted an investigation combining transcriptomics and mass spectrometry to identify its neuropeptides. Specifically, in silico searches of the L. vannamei EST database were conducted to identify putative prepro-hormone-encoding transcripts, with the mature peptides contained within the deduced precursors predicted via online software programs and homology to known isoforms. MALDI-FT mass spectrometry was used to screen tissue fragments and extracts via accurate mass measurements for the predicted peptides, as well as for known ones from other species. ESI-Q-TOF tandem mass spectrometry was used to de novo sequence peptides from tissue extracts. In total 120 peptides were characterized using this combined approach, including 5 identified both by transcriptomics and by mass spectrometry (e.g. pQTFQYSRGWTNamide, Arg7-corazonin, and pQDLDHVFLRFamide, a myosuppressin), 49 predicted via transcriptomics only (e.g. pQIRYHQCYFNPISCF and pQIRYHQCYFIPVSCF, two C-type allatostatins, and RYLPT, authentic proctolin), and 66 identified solely by mass spectrometry (e.g. the orcokinin NFDEIDRAGMGFA). While some of the characterized peptides were known L. vannamei isoforms (e.g. the pyrokinins DFAFSPRLamide and ADFAFNPRLamide), most were novel, either for this species (e.g. pEGFYSQRYamide, an RYamide) or in general (e.g. the tachykinin-related peptides APAGFLGMRamide, APSGFNGMRamide and APSGFLDMRamide). Collectively, our data not only expand greatly the number of known L. vannamei neuropeptides, but also provide a foundation for future investigations of the physiological roles played by them in this commercially important species.
functional genomics; expressed sequence tag (EST); matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS)
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 [2-9]. 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 [2-3]. 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.
FMRFamide-related peptide; neuropeptides; immuno-dot blot screening assay; immunoprecipitation; MALDI-FTMS; ESI-QTOF
In this study we report an improved protocol that combines simplified sample preparation and micro-scale separation for mass spectrometric analysis of neuropeptides from individual neuroendocrine organs of crab Cancer borealis. A simple, one-step extraction method with commonly used matrix-assisted laser desorption/ionization (MALDI) matrix, 2,5-dihydroxybenzoic acid (DHB), in saturated aqueous solution, is employed for improved extraction of neuropeptides. Furthermore, a novel use of DHB as background electrolyte for capillary electrophoresis (CE) separation in the off-line coupling of CE to MALDI-Fourier transform mass spectrometric (FT-MS) detection is also explored. The new CE electrolyte exhibits full compatibility with MALDI-MS analysis of neuropeptides in that both the peptide extraction process and MALDI detection utilize DHB. In addition, enhanced resolving power and improved sensitivity are also observed for CE-MALDI-MS of peptide mixture analysis. Collectively, the use of DHB has simplified the extraction and reduced the sample loss by elimination of homogenizing, drying, and desalting processes. In the mean time, the concurrent use of DHB as CE separation buffer and subsequent MALDI-MS detection offers improved spectral quality by eliminating the interferences from typical CE electrolyte in MALDI detection.
Due to the complexity of the mammalian central nervous system neuropeptidomic studies in mammals often yield very complicated mass spectra that make data analysis difficult. Careful sample preparation and extraction protocols must be employed in order to minimize spectral complexity and enable extraction of useful information on neuropeptides from a given sample. Controlling post-mortem protease activity is essential to simplifying mass spectra and to identifying low-abundance neuropeptides in tissue samples. Post-mortem microwave-irradiation coupled with cryostat dissection has proven to be effective in arresting protease activity to allow detection of endogenous neuropeptides instead of protein degradation products.
Neuropeptide; Extraction; Microwave; Protease activity; Sample preparation; Mass spectrometry; HPLC; Peptide sequencing/identification
The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10−6M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies < 0.7Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.
allatostatin; crustaceans; neuromodulation; stomatogastric nervous system; neuropeptides; peptide sequencing
Carcinus maenas, commonly known as the European green crab, is one of the best-known and most successful marine invasive species. While a variety of natural and anthropogenic mechanisms are responsible for the geographic spread of this crab, its ability to adapt physiologically to a broad range of salinities, temperatures and other environmental factors has enabled successful establishment in these new habitats. To extend our understanding of hormonal control in C. maenas, including factors that allow for its extreme adaptability, we have undertaken a mass spectral/functional genomics investigation of the neuropeptides used by this organism. Via a strategy combining MALDI-based high resolution mass profiling, biochemical derivatization, and nanoscale separation coupled to tandem mass spectrometric sequencing, 122 peptide paracrines/hormones were identified from the C. maenas central nervous system and neuroendocrine organs. These peptides include 31 previously described Carcinus neuropeptides (e.g. NSELINSILGLPKVMNDAamide [β-pigment dispersing hormone] and PFCNAFTGCamide [crustacean cardioactive peptide]), 49 peptides only described in species other than the green crab (e.g. pQTFQYSRGWTNamide [Arg7-corazonin]), and 42 new peptides de novo sequenced here for the first time (e.g. the pyrokinins TSFAFSPRLamide and DTGFAFSPRLamide). Of particular note are a collection of 25 FMRFamide-like peptides (including 9 new isoforms sequenced de novo) and a collection of 25 A-type allatostatin peptides (including 10 new sequences reported for the first time) in this study. Both peptide families are among the most diverse families, each containing a large number of isoforms in arthropod species. Also of interest was the identification of two SIFamide isoforms, GYRKPPFNGSIFamide and VYRKPPFNGSIFamide, the latter peptide known previously only from members of the astacidean genus Homarus. Using transcriptome analyses, 15 additional peptides were characterized, including an isoform of bursicon β and a neuroparsin-like peptide. Collectively, the data presented in this study not only greatly expand the number of identified C. maenas neuropeptides, but also provide a framework for future investigations of the physiological roles played by these molecules in this highly adaptable species.
Carcinus maenas; European green crab; matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS); electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS); expressed sequence tag (EST); tblastn; blastp; transcriptomics; neuropeptide; neurohormone; peptide sequencing; supraoesophageal ganglion; brain; thoracic ganglia; sinus gland (SG); pericardial organ (PO)