Isocratic reverse-phase HPLC separation and RIA used in the analysis of neuropeptides in brain tissue (original) (raw)
Related papers
2020
Neuropeptides are the largest and most diverse class of cell-cell signaling molecules in the brain. They are expressed and synthesized by neurons and endocrine organs, released upon stimulation and act by binding to specific cell surface receptors that initiate a cascade of downstream signaling mechanisms. Compelling evidence from several previous studies has demonstrated their role in several physiological functions such as appetite regulation, nociception, locomotion, reproduction, learning and memory. Given their important role as the molecular messengers of a biological system, there is a lot of interest in the accurate identification and characterization of these peptides. However, the task of characterizing them comes with several intrinsic challenges. First, these peptides undergo rapid post-mortem degradation during the extraction and analysis phase. Measuring depleted levels of peptides from a vast pool of ubiquitous peptides and degraded proteins requires unique sampling a...
High Identification Rates of Endogenous Neuropeptides from Mouse Brain
Journal of Proteome Research, 2012
Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC−MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.
Chromatographia, 2004
A method for determination of some biologically active penta-and nona-peptides under isocratic conditions in capillary liquid chromatography was developed. Separation system consisting of XTerra C18 stationary phase and mobile phase composed of a mixture of acetonitrile with 0.1% trifluoroacetic acid (TFA) and water with 0.1% TFA in the ratios 75/25 (v/v) and 85/ 15 (v/v) was suitable not only for a good resolution of enkephalin and vasopressin related peptides, respectively, but it also enabled separation of the respective biopeptides from other constituents of human urine. Calibration curves for the studied peptides were linear in the measured concentration range from 1.00 to 1.57·10 )2 mg mL )1 . The limit of detection and limit of quantification were in the range of units of lg mL )1 and tens of lg mL )1 , respectively; slightly higher values were obtained for nonapeptides. Determination of certain biologically active peptides in urine can serve in future as a tool for diagnosis of various diseases, e.g. autism.
Optimisation of high performance liquid chromatography separation of neuroprotective peptides
Journal of Chromatography A, 2005
The study of experimental design conjunction with artificial neural networks for optimisation of isocratic ion-pair reverse phase HPLC separation of neuroprotective peptides is reported. Different types of experimental designs (full-factorial, fractional) were studied as suitable input and output data sources for ANN training and examined on mixtures of humanin derivatives. The independent input variables were: composition of mobile phase, including its pH, and column temperature. In case of a simple mixture of two peptides, the retention time of the most retentive component and resolution were used as the dependent variables (outputs). In case of a complex mixture with unknown number of components, number of peaks, sum of resolutions and retention time of ultimate peak were considered as output variables. Fractional factorial experimental design has been proved to produce sufficient input data for ANN approximation and thus further allowed decreasing the number of experiments necessary for optimisation. After the optimal separation conditions were found, fractions with peptides were collected and their analysis using off-line matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was performed.
Analytical Chemistry, 1980
Experimental conditions and parameters involved in high performance liquid chromatographic separations of seven neurohypophyseal hormones on several reverse phase columns were investigated. These peptides Include arginine vasotocin, lysine vasopressin, arginine vasopressin, mesotocin, Isotocin, oxytocin, and glumltocln. The effects of carbon chain length of the reverse phase support and organic solvent were examined. Using the appropriate solvent system and column, all of the peptides were separated from one another. Separation of the peptides was only part of the goal. The beginning of a study to understand the Interaction of the peptides with the stationary phase as a function of structure was also undertaken. This study led to the conclusion that the major parameters which allow effective separation of these structurally and conformationally closely related peptides are both the eluting strength of the mobile phase and the chemical composition of the stationary phase.
The absolute quantification of endogenous levels of brain neuropeptides in vivo using LC–MS/MS
Bioanalysis, 2011
Neuropeptides represent a large group of neuroactive messengers present in both the CNS and the peripheral nervous system [1]. These peptides act as neurotransmitters or neuromodulators, growth factors, neurohormones and/or modulators of the immune system [2-5]. Compared with proteins, neuropeptides are smaller (3-100 amino acid residues long) and they have a less complex 3D structure. In comparison with the classical monoamine and amino acid neurotransmitters, they are up to 50-times larger and their brain concentrations are much lower [2]. Neuropeptides do not directly interfere with the normal, fast synaptic transmission, but they have slow modulating effects on the classical neurotransmitters [2,6]. Neuropeptides are involved in several physiological functions, including learning and memory, pain and anxiety, appetite control, stress and social behavior [1]. They also have a role in pathological processes such as addiction, depression, autism and epilepsy. Therefore, there is much interest in these endogenous modulators and their receptors as possible new drug targets. Renowned researchers in the field of epilepsy have, for example, selected neuropeptides as one of the most promising areas for development of new antiepileptic drugs with original mechanisms of action [7,8]. First, neuropeptides are preferentially released when neurons fire at high frequencies, which typically happens during epileptic seizures. Second, in most cases, they co-exist with the classical neurotransmitters, but they do not participate in fast synaptic transmission [2,8,9]. For these reasons, pharmacotherapeutical application of peptidomimetics could be less prone to side effects as compared with drugs that act on the classical fast-acting neurotransmitters [10]. Mechanism of neuropeptide release Generally, neuropeptides are synthesized at the ribosomes in the cell bodies of neurons, and to a lesser extent, also in the dendrites as large, biologically inert precursor proteins. In addition, production of peptides can also occur in glial cells. The prepropeptides subsequently undergo cleavage and post-translational modifications to generate the active peptide. Hereafter, they are packed in large dense-core vesicles (LDCVs) and transported into the axons and dendrites. LDCVs differ from small synaptic vesicles, the predominant vesicle for release of the classical neurotransmitters, in size, physical density and in composition of the membrane proteins involved in the release processes (Figure 1). Only a small fraction (~1%) of the LDCVs present at the release site are available for release after depolarization. In addition, all peptide release appears to be Ca 2+ dependent and with high Ca 2+ affinity. The latter is necessary since the LDCVs are spatially separated from the Ca 2+ channels. This all results in latencies of LDCV release ranging from 30 to 2000 ms. Typically, neuropeptides are subsequently only released after strong stimulation, such as high frequency firing or burst-patterned The absolute quantification of endogenous levels of brain neuropeptides in vivo using LC-MS/MS Neuropeptides seem to play an important role when the CNS is challenged. In order to obtain better insights into the central peptidergic effects, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano-LC-MS/MS methods. A number of LC-MS/MS and microdialysis parameters need to be optimized to achieve maximal sensitivity. The optimized and validated methods can be used to investigate the in vivo neuropeptide release during pathological conditions, in this way initiating new and immense challenges for the development of new drugs.
ELECTROPHORESIS, 2008
In this study, the suitability of SPE coupled on-line to CE-electrospray-MS (SPE-CE-ESI-MS) was evaluated for the analysis of neuropeptides in human plasma. First, CE-ESI-MS was investigated and a sample pretreatment based on precipitation with ACN was used for cleanup of plasma samples. The main quality parameters were determined and were consistent with those previously obtained for the analysis of standard mixtures, e.g., the LODs were of around 1 mg/mL. SPE-CE-ESI-MS employing microcartridges containing a C18 stationary phase was explored in order to decrease the LODs. A double-step sample cleanup pretreatment consisting of precipitation with ACN and ultrafiltration through 3000 Da MW cut-off membranes was needed to prevent the microcartridge saturation. Repeatability, reproducibility, linearity, and LODs of the SPE-CE-ESI-MS method and the durability of the microcartridges were studied. The LODs were markedly improved, ranging between 10 and 0.1 ng/mL depending on the peptide.
Journal of Proteome Research, 2009
In recent years, mass spectrometry (MS) based techniques have made their entrance in the analysis of endogenous peptides extracted from nervous tissue. In this study, we introduce a novel peptide extraction procedure using 8 M urea, next to the more established extraction method that uses acetic acid. The extracted peptide mixtures are analyzed by both high-resolution nanoLC MS/MS using collision induced dissociation (CID) on an LTQ-Orbitrap and nanoLC electron transfer induced dissociation (ETD) on a linear ion trap. The combined use of the two extraction methods significantly increased the yield of identified endogenous neuropeptides. The multiplexed use of high mass accuracy mass spectrometry and the ETD fragmentation technique further increased the yield and confidence of peptide identifications. Furthermore, reduction of disulfide bridges during sample preparation was essential in the identification of several endogenous peptides containing cysteine disulfide bonds. Through this study, we identified in total 142 peptides in extracts of the mouse pituitary tissue, whereby 43 uniquely in the urea extract and 11 uniquely in the acetic acid extract. A large number of detected endogenous peptides were reported previously, but we confidently identified 22 unreported peptides that possess characteristics of endogenous peptides and are thus interesting targets to be explored further.
Analytical Chemistry, 2010
Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides prepared from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using the SRM mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in whole mouse brain matrix are demonstrated, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in μLC-MS/MS based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification.
2009
In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies dcscribed here usc differcnt extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorptionlionization (MALDI) and eleetrospray ionization (ESl). Thesc strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectromctric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, includin...