Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway.
NK cells; IL-2 signaling; Mass spectrometry pathways; Proteomics; Lab-free quantification; Multi-dimensional separation
(RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) is a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and is routinely used with rodent models to investigate the role of NMDA receptors in brain function. This highly polar compound is difficult to separate from biological matrices. A reliable and sensitive assay was developed for the determination of CPP in plasma and tissue. In order to overcome the challenges relating to the physicochemical properties of CPP we employed an initial separation using solid phase extraction harnessing mixed-mode anion exchange. Then an ion-pair UPLC C18 separation was performed followed by MS/MS with a Waters Acquity UPLC interfaced to an AB Sciex QTrap 5500 mass spectrometer, which was operated in positive ion ESI mode. Multiple reaction monitoring (MRM) mode was utilized to detect the analyte and internal standard. The precursor to product ions used for quantitation for CPP and internal standard were m/z 252.958 → 207.100 and 334.955 → 136.033, respectively. This method was applied to a pharmacokinetic study and examined brain tissue and plasma concentrations following intravenous and intraperitoneal injections of CPP. The elimination half-life (t1/2) of CPP was 8.8 minutes in plasma and 14.3 minutes in brain tissue, and the plasma to brain concentration ratio was about 18:1. This pharmacokinetic data will aid the interpretation of the vast number of studies using CPP to investigate NMDA receptor function in rodents and the method itself can be used to study many other highly polar analytes of interest.
We synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addition, the dTPT3 scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3PA-dNaM pair, which is amplified only slightly less well. The identification of dTPT3 represents significant progress towards developing an unnatural base pair for the in vivo expansion of an organism's genetic alphabet and for a variety of in vitro biotechnology applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.
Mass spectrometric imaging (MSI) is a powerful tool that grants the ability to investigate a broad mass range of molecules, from small molecules to large proteins, by creating detailed distribution maps of selected compounds. To date, MSI has demonstrated its versatility in the study of neurotransmitters and neuropeptides of different classes toward investigation of neurobiological functions and diseases. These studies have provided significant insight in neurobiology over the years and current technical advances are facilitating further improvements in this field. neurotransmitters, focusing specifically on the challenges and recent Herein, we advances of MSI of neurotransmitters.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has been employed as a detection method for both capillary electrophoresis (CE)-MALDI and liquid chromatography (LC)-MALDI analysis. Based on our previous studies, here we report a new interface to couple LC to MSI by employing an automated matrix sprayer. The LC trace is directly collected on a ground stainless steel MALDI plate and dried. The matrix is sprayed on the MALDI plate using a programmable matrix sprayer. With the highly uniform matrix layers produced from the sprayer, MS image signal quality is significantly improved with enhanced signal-to-noise ratios for analyte peaks. With the programmable matrix application and imaging MS data acquisition, the new LC-MSI platform exhibits highly stable and reproducible performance. A total of 87 bovine serum albumin (BSA) tryptic peptides and 295 putative neuropeptides from blue crab pericardial organs have been observed with LC-MSI analysis, exhibiting better performance in terms of peptide coverage than regular LC-MALDI with discrete spot collection and our previously reported LC-MSI interface with matrix delivered by a capillary. In addition to relative quantitation with isotopic labeling as we previously demonstrated, we performed the first absolute quantitation using the new LC-MSI platform and obtained accurate quantitation results for neuropeptides, indicating great potential for quantitative analysis of complex samples.
The conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2 kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems.
Neuropeptide; peptidomics; de novo sequencing; mass spectrometry; crustacean
spatial localization and molecular distribution of metabolites and
neurotransmitters within biological organisms is of tremendous interest
to neuroscientists. In comparison to conventional imaging techniques
such as immunohistochemistry, matrix-assisted laser desorption/ionization
(MALDI) mass spectrometric imaging (MSI) has demonstrated its unique
advantage by directly localizing the distribution of a wide range
of biomolecules simultaneously from a tissue specimen. Although MALDI-MSI
of metabolites and neurotransmitters is hindered by numerous matrix-derived
peaks, high-resolution and high-accuracy mass spectrometers (HRMS)
allow differentiation of endogenous analytes from matrix peaks, unambiguously
obtaining biomolecular distributions. In this study, we present MSI
of metabolites and neurotransmitters in rodent and crustacean central
nervous systems acquired on HRMS. Results were compared with those
obtained from a medium-resolution mass spectrometer (MRMS), tandem
time-of-flight instrument, to demonstrate the power and unique advantages
of HRMSI and reveal how this new tool would benefit molecular imaging
applications in neuroscience.
imaging; high spectral resolution; metabolites; neurotransmitters; central nervous system; matrix-assisted laser desorption/ionization
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant and teratogen that produces cardiac toxicity in the developing zebrafish. Here we adopted a label free quantitative proteomic approach based on normalized spectral abundance factor (NSAF) to investigate the disturbance of the cardiac proteome induced by TCDD in the adult zebrafish heart. The protein expression level changes between heart samples from TCDD treated and control zebrafish were systematically evaluated by a large scale MudPIT analysis which incorporated triplicate analyses for both control and TCDD exposed heart proteomic samples to overcome the data dependant variation in shotgun proteomic experiments and obtain a statistically significant protein dataset with improved quantification confidence. A total of 519 and 443 proteins were identified in hearts collected from control and TCDD treated zebrafish, respectively, among which 106 proteins showed statistically significant expression changes. After correcting for the experimental variation between replicate analyses by statistical evaluation, 55 proteins exhibited NSAF ratio above 2 and 43 proteins displayed NSAF ratio smaller than 0.5, with statistical significance by t-test (p < 0.05). The proteins identified as altered by TCDD encompass a wide range of biological functions including calcium handling, myocardium cell architecture, energy production and metabolism, mitochondrial homeostasis, and stress response. Collectively, our results indicate that TCDD exposure alters the adult zebrafish heart in a way that could result in cardiac hypertrophy and heart failure, and suggests a potential mechanism for the diastolic dysfunction observed in TCDD exposed embryos.
TCDD; cardiac toxicity; zebrafish; heart proteomics; mass spectrometry; spectral counting; quantitation
Mass spectrometric imaging (MSI) has rapidly increased its presence in the pharmaceutical sciences. While quantitative whole-body autoradiography and microautoradiography are the traditional techniques for molecular imaging of drug delivery and metabolism, MSI provides advantageous specificity that can distinguish the parent drug from metabolites and modified endogenous molecules. This review begins with the fundamentals of MSI sample preparation/ionization, and then moves on to both qualitative and quantitative applications with special emphasis on drug discovery and delivery. Cutting-edge investigations on sub-cellular imaging and endogenous signaling peptides are also highlighted, followed by perspectives on emerging technology and the path for MSI to become a routine analysis technique.
ADME; spatial distribution; quantitative mass spectrometry; PK/PD; molecular imaging; pharmaceuticals
A semi-automated analytical platform featuring the coupling of monolithic reversed-phase liquid chromatography (RPLC) to matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) has been developed and evaluated. This is the first time that LC separation is readily coupled to MS imaging detection for the analysis of complex peptide mixtures both qualitatively and quantitatively. Methacrylate-based monolithic column with C12 functional groups is fabricated for fast RPLC separation. The LC flow and matrix flow are collected on a commercially available MALDI plate which is mechanically controlled and analyzed with MALDI MSI subsequently. Both tryptic peptides digested from bovine serum albumin (BSA) and endogenous neuropeptides extracted from the blue crab Callinectes sapidus are analyzed with this novel LC–MSI platform. Compared with regular offline LC fractionation coupled with MALDI MS detection, LC–MSI exhibits significantly increased MS signal intensity due to retaining of temporal resolution from separation dimension via continuous sampling, which results in increased number of peptides detected and accurate quantitation. In addition, imaging signals enable improved data analysis based on either mass-to-charge ratio or retention time, which is extremely beneficial for the analysis of complex analytes. These findings have demonstrated the potential of employing LC–MSI platform for enhanced proteomics and peptidomics studies.
Liquid chromatography; Mass spectrometric imaging; LC; MSI; Monolith; Neuropeptides; Quantitation
Gambogic acid (GA), the main active component of gamboge resin, has potent antitumor activity both in vivo and in vitro. However, the underlying molecular mechanisms remain unclear. In this study, we found that GA could initiate autophagy in colorectal cancer cells, and inhibition of the autophagy process accelerated the effect of proliferative inhibition and apoptotic cell death induced by GA, implying a protective role of autophagy. Two-dimensional electrophoresis-based proteomics showed that GA treatment altered the expression of multiple proteins involved in redox signaling and lipid metabolism. Functional studies revealed that GA-induced dysregulation of lipid metabolism could activate 5-lipoxygenase (5-LOX), resulting in intracellular ROS accumulation, followed by inhibition of Akt-mTOR signaling and autophagy initiation. Finally, results using a xenograft model suggested ROS-induced autophagy protect against the antitumor effect of GA. Taken together, these data showed new biological activities of GA against colorectal cancer underlying the protective role of ROS-induced autophagy. This study will provide valuable insights for future studies regarding the anticancer mechanisms of GA.
Mass spectrometry imaging (MSI) is a powerful tool that grants the ability to investigate a broad mass range of molecules from small molecules to large proteins by creating detailed distribution maps of selected compounds. Its usefulness in biomarker discovery towards clinical applications has obtained success by correlating the molecular expression of tissues acquired from MSI with well-established histology.
To date, MSI has demonstrated its versatility in clinical applications, such as biomarker diagnostics of different diseases, prognostics of disease severities and metabolic response to drug treatment, etc. These studies have provided significant insight in clinical studies over the years and current technical advances are further facilitating the improvement of this field. Although the underlying concept is simple, factors such as choice of ionization method, sample preparation, instrumentation and data analysis must be taken into account for successful applications of MSI. Herein, we briefly reviewed these key elements yet focused on the clinical applications of MSI that cannot be addressed by other means.
Challenges and future perspectives in this field are also discussed to conclude that the ever-growing applications with continuous development of this powerful analytical tool will lead to a better understanding of the biology of diseases and improvements in clinical diagnostics.
Mass spectrometry imaging; MALDI; DESI; clinical; biomarkers
Traditionally, the d-amino
acid containing peptide (DAACP) candidate can be discovered by observing
the differences of biological activity and chromatographic retention
time between the synthetic peptides and naturally occurring peptides.
However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel
site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis
of mass spectrometry (MS)-generated epimeric fragment ions. Briefly,
the d/l-peptide epimers were separated by online
reversed-phase liquid chromatography and fragmented by collision-induced
dissociation (CID), followed by IMS analysis. The epimeric fragment
ions resulting from d/l-peptide epimers exhibit
conformational differences, thus showing different mobilities in IMS.
The arrival time shift between the epimeric fragment ions was used
as criteria to localize the d-amino acid substitution. The
utility of this strategy was demonstrated by analysis of peptide epimers
with different molecular sizes, [d-Trp]-melanocyte-stimulating
hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I,
and their counterparts that contain all-l amino acids. Furthermore,
the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated
from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down
sequencing approaches. The
IMS data acquired using our novel site-specific strategy localized
the site of isomerization of l- to d-Phe at the
third residue of the CHHs from the N-terminus. Collectively, this
study demonstrates a new method for discovery of DAACPs using IMS
technique with the ability to localize d-amino acid residues.
In the developing mammalian brain, inhibition of NMDA receptor can induce widespread neuroapoptosis, inhibit neurogenesis and cause impairment of learning and memory. Although some mechanistic insights into adverse neurological actions of these NMDA receptor antagonists exist, our understanding of the full spectrum of developmental events affected by early exposure to these chemical agents in the brain is still limited. Here we attempt to gain insights into the impact of pharmacologically induced excitatory/inhibitory imbalance in infancy on the brain proteome using mass spectrometric imaging (MSI). Our goal was to study changes in protein expression in postnatal day 10 (P10) rat brains following neonatal exposure to the NMDA receptor antagonist dizocilpine (MK801). Analysis of rat brains exposed to vehicle or MK801 and comparison of their MALDI MS images revealed differential relative abundances of several proteins. We then identified these markers such as ubiquitin, purkinje cell protein 4 (PEP-19), cytochrome c oxidase subunits and calmodulin, by a combination of reversed-phase (RP) HPLC fractionation and top-down tandem MS platform. More in-depth large scale study along with validation experiments will be carried out in the future. Overall, our findings indicate that a brief neonatal exposure to a compound that alters excitatory/inhibitory balance in the brain has a long term effect on protein expression patterns during subsequent development, highlighting the utility of MALDI-MSI as a discovery tool for potential biomarkers.
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