The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology. Proteomics technologies have evolved to produce large datasets of proteins or peptides involved in various biological and disease progression processes producing testable hypothesis for complex biological questions. This review provides an introduction and insight to relevant topics in proteomics and peptidomics including biological material selection, sample preparation, separation techniques, peptide fragmentation, post-translation modifications, quantification, bioinformatics, and biomarker discovery and validation. In addition, current literature and remaining challenges and emerging technologies for proteomics and peptidomics are presented.
Mass spectrometry; proteomics; peptidomics; review; post-translation modifications; fragmentation; separation; protein quantitation; sample preparation; biomarker discovery; validation; bioinformatics
Tissue heat stabilization is a vital component in successful mammalian neuropeptidomic studies. Heat stabilization using focused microwave irradiation, conventional microwave irradiation, boiling, and treatment with the Denator Stabilizor T1 have all proven effective in arresting post-mortem protein degradation. Although research has reported the presence of protein fragments in crustacean hemolymph when protease inhibitors were not added to the sample, the degree to which postmortem protease activity affects neuropeptidomic tissue studies in crustacean species has not been investigated in depth. This work examines the need for Stabilizor T1 or boiling tissue stabilization methods for neuropeptide studies of Callinectes sapidus (blue crab) pericardial organ tissue. Neuropeptides in stabilized and non-stabilized tissue are extracted using acidified methanol or N,N-Dimethylformamide (DMF) and analyzed by MALDI-TOF and nanoLC-ESI-MS/MS platforms. Post-mortem fragments did not significantly affect MALDI analysis in the range m/z 650–1600, but observations in ESI MS/MS experiments suggest that putative post-mortem fragments can mask neuropeptide signal and add spectral complexity to crustacean neuropeptidomic studies. The impact of the added spectral complexity did not dramatically affect the number of detected neuropeptides between stabilized and non-stabilized tissues. However, it is prudent that neuropeptidomic studies of crustacean species include a preliminary experiment using the heat stabilization method to assess the extent of neuropeptide masking by larger, highly charged molecular species.
Neuropeptide; crustacean; Callinectes sapidus; heat stabilization; DiLeu; MALDI; ESI; peptidomics; Stabilizor T1; post-mortem
Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in direct contact with the brain. A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the low volumes of CSF that are obtainable from mice. As a model system in which to test this approach, we utilized transgenic mice that over-express the intermediate filament glial fibrillary acidic protein (GFAP). These mice are models for Alexander disease (AxD), a severe leukodystrophy in humans. From the CSF of control and transgenic mice we report the identification of 289 proteins, with relative quantification of 103 proteins. Biological and technical triplicates were performed to address animal variability as well as reproducibility in mass spectrometric analysis. Relative quantitation was performed using distributive normalized spectral abundance factor (dNSAF) spectral counting analysis. A panel of biomarker proteins with significant changes in the CSF of GFAP transgenic mice has been identified with validation from ELISA and microarray data, demonstrating the utility of our methodology and providing interesting targets for future investigations on the molecular and pathological aspects of AxD.
Cerebrospinal fluid; proteomics; spectral counting; Alexander disease; mass spectrometry; immunodepletion; transgenic; GFAP overexpressor
Nanostructure-initiator mass spectrometry (NIMS) is a recently developed matrix-free laser desorption/ionization technique that has shown promise for peptide analyses. It is also useful in mass spectrometric imaging (MSI) studies of small molecule drugs, metabolites, and lipids, minimizing analyte diffusion caused by matrix application. In this study, NIMS and matrix-assisted laser desorption/ionization (MALDI) MSI of a crustacean model organism Cancer borealis brain were compared. MALDI was found to perform better than NIMS in these neuropeptide imaging experiments. Twelve neuropeptides were identified in MALDI MSI experiments whereas none were identified in NIMS MSI experiments. In addition, lipid profiles were compared using each ionization method. Both techniques provided similar lipid profiles in the m/z range 700 – 900.
Microdialysis (MD) is a useful sampling tool for many applications due to its ability to permit sampling from an animal concurrent with normal activity. MD is of particular importance in the field of neuroscience, in which it is used to sample neurotransmitters (NTs) while the animal is behaving in order to correlate dynamic changes in NTs with behavior. One important class of signaling molecules, the neuropeptides (NPs), however, presented significant challenges when studied with MD, due to the low relative recovery (RR) of NPs by this technique. Affinity-enhanced microdialysis (AE-MD) has previously been used to improve recovery of NPs and similar molecules. For AE-MD, an affinity agent (AA), such as an antibody-coated particle or free antibody, is added to the liquid perfusing the MD probe. This AA provides an additional mass transport driving force for analyte to pass through the dialysis membrane, and thus increases the RR. In this work, a variety of AAs have been investigated for AE-MD of NPs in vitro and in vivo, including particles with C18 surface functionality and antibody-coated particles. Antibody-coated magnetic nanoparticles (AbMnP) provided the best RR enhancement in vitro, with statistically significant (p<0.05) enhancements for 4 out of 6 NP standards tested, and RR increases up to 41-fold. These particles were then used for in vivo MD in the Jonah crab, Cancer borealis, during a feeding study, with mass spectrometric (MS) detection. 31 NPs were detected in a 30 min collection sample, compared to 17 when no AA was used. The use of AbMnP also increased the temporal resolution from 4–18 hrs in previous studies to just 30 min in this study. The levels of NPs detected were also sufficient for reliable quantitation with the MS system in use, permitting quantitative analysis of the concentration changes for 7 identified NPs on a 30 min time course during feeding.
Microdialysis; In Vivo measurement; Affinity-enhanced microdialysis; Neuropeptides; Mass spectrometry; LC-MS; LC-ESI-QTOF; Crustacean; Hemolymph
Considerable effort has been devoted to characterizing the crustacean stomatogastric nervous system (STNS) with great emphasis on comprehensive analysis and mapping distribution of its diverse neuropeptide complement. Previously, immunohistochemistry (IHC) has been applied to this endeavor yet with identification accuracy and throughput compromised. Therefore, molecular imaging methods are pursued to unequivocally determine the identity and location of the neuropeptides at a high spatial resolution. In this work, we developed a novel multi-faceted mass spectrometric strategy combining profiling and imaging techniques to characterize and map neuropeptides from the blue crab Callinectes sapidus STNS at the network level. In total, 55 neuropeptides from 10 families were identified from the major ganglia in the C. sapidus STNS for the first time, including the stomatogastric ganglion (STG), the paired commissural ganglia (CoG), the esophageal ganglion (OG), and the connecting nerve stomatogastric nerve (stn) using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) and the MS/MS capability of this technique. In addition, the locations of multiple neuropeptides were documented at a spatial resolution of 25 μm in the STG and upstream nerve using MALDI-TOF/TOF and high-mass-resolution and high-mass-accuracy MALDI-Fourier transform ion cyclotron resonance (FT-ICR) instrument. Furthermore, distributions of neuropeptides in the whole C. sapidus STNS were examined by imaging mass spectrometry (IMS). Different isoforms from the same family were simultaneously and unambiguously mapped, facilitating the functional exploration of neuropeptides present in the crustacean STNS and exemplifying the revolutionary role of this novel platform in neuronal network studies.
Crustacean; STNS; Neuropeptide; Imaging mass spectrometry; High resolution MS
Dopamine (DA) can produce divergent effects at different time scales. DA has opposing immediate and long-term effects on the transient potassium current (IA) within neurons of the pyloric network, in the Panulirus interruptus stomatogastric ganglion. The lateral pyloric neuron (LP) expresses type 1 DA receptors (D1Rs). A 10 min application of 5-100 μM DA decreases LP IA by producing a decrease in IA maximal conductance (Gmax) and a depolarizing shift in IA voltage dependence through a cAMP-Protein kinase A (PKA) dependent mechanism. Alternatively, a 1 hr application of DA (≥5 nM) generates a persistent (measured 4 hr after DA washout) increase in IA Gmax in the same neuron, through a mechanistic target of rapamycin (mTOR) dependent translational mechanism. We examined the dose, time and protein dependencies of the persistent DA effect.
We found that disrupting normal modulatory tone decreased LP IA. Addition of 500 pM-5 nM DA to the saline for 1 hr prevented this decrease, and in the case of a 5 nM DA application, the effect was sustained for >4 hrs after DA removal. To determine if increased cAMP mediated the persistent effect of 5nM DA, we applied the cAMP analog, 8-bromo-cAMP alone or with rapamycin for 1 hr, followed by wash and TEVC. 8-bromo-cAMP induced an increase in IA Gmax, which was blocked by rapamycin. Next we tested the roles of PKA and guanine exchange factor protein activated by cAMP (ePACs) in the DA-induced persistent change in IA using the PKA specific antagonist Rp-cAMP and the ePAC specific agonist 8-pCPT-2′-O-Me-cAMP. The PKA antagonist blocked the DA induced increases in LP IA Gmax, whereas the ePAC agonist did not induce an increase in LP IA Gmax. Finally we tested whether extracellular signal regulated kinase (Erk) activity was necessary for the persistent effect by co-application of Erk antagonists PD98059 or U0126 with DA. Erk antagonism blocked the DA induced persistent increase in LP IA.
These data suggest that dopaminergic tone regulates ion channel density in a concentration and time dependent manner. The D1R- PKA axis, along with Erk and mTOR are necessary for the persistent increase in LP IA induced by high affinity D1Rs.
Herein we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogenous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultra-high mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling four to six-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that, it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.
capillary electrophoresis; CE; PACE; MALDI; mass spectrometric imaging; MSI; peptides; neuropeptides; quantitation
Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiological agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at sub-femtomole levels while animal bioassays, cell culture, and in vitro conversion assays offer ultrasensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein’s signature peptide, often with sub-femtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors’ knowledge, this is the first report of the use of a non-tryptic peptide in a LC-MRM AQUA workflow.
Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS could revolutionize neuronal network and biomarker discovery studies.
Imaging mass spectrometry; MALDI; neurons; neuroendocrine tissues; sampling; neuropeptides
The blue crab Callinectes sapidus has been used as an experimental model organism for the study of regulation of cardiac activity and other physiological processes. Moreover, it is an economically and ecologically important crustacean species. However, there was no previous report on the characterization of its neuropeptidome. To fill in this gap, we employed multiple sample preparation methods including direct tissue profiling, crude tissue extraction and tissue extract fractionation by HPLC to obtain a complete description of the neuropeptidome of C. sapidus. Matrix-assisted laser desorption/ionization (MALDI)-Fourier transform mass spectrometry (FTMS) and MALDI-time-of-flight (TOF)/TOF were utilized initially to obtain a quick snapshot of the neuropeptide profile, and subsequently nanoflow liquid chromatography (nanoLC) coupled with electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem MS analysis of neuropeptide extracts was conducted for de novo sequencing. Simultaneously, the pericardial organ (PO) tissue extract was labeled by a novel N, N-dimethylated leucine (DiLeu) reagent, offering enhanced fragmentation efficiency of peptides. In total, 130 peptide sequences belonging to 11 known neuropeptide families including orcomyotropin, pyrokinin, allatostatin A (AST-A), allatostatin B (AST-B), FMRFamide-like peptides (FLPs), and orcokinin were identified. Among these 130 sequences, 44 are novel peptides and 86 are previously identified. Overall, our results lay the groundwork for future physiological studies of neuropeptides in C. sapidus and other crustaceans.
Callinectes sapidus; pericardial organ; de novo sequencing; neuropeptidome; neuropeptides; chemical derivatization
This work investigates the suitability of molecular weight cut-off membrane-based centrifugal filter devices (MWCO) for sub-microgram peptide enrichment passing through the membrane by introduction of methanol and a salt modifier. Using a neuropeptide standard, bradykinin, a reduction in sample loss of over two orders of magnitude is demonstrated with and without undigested protein present. Additionally, a bovine serum albumin (BSA) tryptic digestion was investigated and 27 tryptic peptides were identified using MALDI mass spectrometry whereas only two BSA tryptic peptides were identified after MWCO separation using H2O. The protocol presented here enhances recovery from MWCO separation for sub-μg peptide samples.
Mass spectrometry; Peptides; Molecular weight cut-off fractionation; Sample preparation
The stomatogastric nervous system (STNS) of the American
lobster Homarus americanus serves as a useful model
of neuromodulatory substances such as peptides and their roles in
the generation of rhythmic behaviors. As a central component of the
STNS, the stomatogastric ganglion (STG) is rich in neuropeptides and
contains well-defined networks of neurons, serving as an excellent
model system to study the effect of neuropeptides on the maturation
of neural circuits. Here, we utilize multiple mass spectrometry (MS)-based
techniques to study the neuropeptide content and abundance in the
STG tissue as related to the developmental stage of the animal. Capillary
electrophoresis (CE)-MS was employed to unambiguously identify low
abundance neuropeptide complements, which were not fully addressed
using previous methods. In total, 35 neuropeptides from 7 different
families were detected in the tissue samples. Notably, 10 neuropeptides
have been reported for the first time in this study. In addition,
we utilized a relative quantitation method to compare neuropeptidomic
expression at different developmental stages and observed sequential
appearance of several neuropeptides. Multiple isoforms within the
same peptide family tend to show similar trends of changes in relative
abundance during development. We also determined that the relative
abundances of tachykinin peptides increase as the lobster grows, suggesting
that the maturation of circuit output may be influenced by the change
of neuromodulatory input into the STG. Collectively, this study expands
our knowledge about neuropeptides in the crustacean STNS and provides
useful information about neuropeptide expression in the maturation
Homarus americanus; matrix-assisted
laser desorption/ionization time-of-flight/time-of-flight mass spectrometry
(MALDI TOF/TOF MS); capillary electrophoresis (CE); neuropeptide; stomatogastric ganglion (STG); stomatogastric
nervous system (STNS); development
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.