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Direct analysis of neuropeptides by in situ MALDI-TOF mass spectrometry in the rat brain
Neuro endocrinology letters
The measure of neuropeptides is an important tool in biology to better define endocrine and neuroendocrine function. Traditionally most methods have relied on the development of specific antibodies. Newer molecular methodologies have used measures of gene expression of neuropeptide precursors, such as Northern blot, PCR or in situ hybridization analysis. Matrix-assisted laser desorption/ionization (MALDI) mass analysis is a novel powerful technique for investigation of neuropeptides. Multiple peptides and peptide forms can be detected simultaneously and with great sensitivity in tissue extracts or partially purified samples. We have now adapted a MALDI methodology for the direct measurement of neuropeptides on fresh tissue sections of rat brains. We have validated the method by examining peptidergic mass profiles of the supraoptic nucleus (SON) and caudate putamen hypothalamic regions. Interestingly, mass profiles showed that vasopressin, which is specifically present in the SON, is...
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.
Neuropeptidomics: Mass spectrometry-based qualitative and quantitative analysis
Methods in Molecular Biology, 2011
Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of signaling molecules that are involved in regulatory functions and behavioral control in the nervous system. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally, and chemically heterogeneous, making them difficult to predict in silico from genomic information. Mature neuropeptides are produced from intricate enzymatic processing of precursor proteins/prohormones via a range of posttranslational modifications, resulting in multiple final peptide products from each prohormone gene. Although there are several methods for targeted peptide studies, mass spectrometry (MS), with its qualitative and quantitative capabilities, is ideally suited to the task. MS provides fast, sensitive, accurate, and highthroughput peptidomic analysis of neuropeptides without requiring prior knowledge of the peptide sequences. Aided by liquid chromatography (LC) separations and bioinformatics, MS is quickly becoming a leading technique in neuropeptidomics. This chapter describes several LC-MS analytical methods to identify, characterize, and quantify neuropeptides while emphasizing the sample preparation steps so integral to experimental success.
Neuropeptides : methods and protocols
2011
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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...
From Genome to Physiology, 2016
MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nature Methods, 4, 828-833. [The state of MALDI mass spectrometry as an information-rich imaging technique with broad current and future application possibilities.]
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.
Neuroendocrinology and its Quantitative Development: A Bioengineering View
BioMedical Engineering OnLine, 2010
Biomedical engineering is clearly present in modern neuroendocrinology, and indeed has come to embrace it in many respects. First, we briefly review the origins of endocrinology until neuroendocrinology, after a long saga, was established in the 1950's decade with quantified results made possible by the radioimmunoassay technique (RIA), a development contributed by the physical sciences. However, instrumentation was only one face of the quantification process, for mathematical models aiding in the study of negative feedback loops, first rather shyly and now at a growing rate, became means building the edifice of mathematical neuroendocrinology while computer assisted techniques help unravel the associated genetic aspects or the nature itself of endocrine bursts by numerical deconvolution analysis. To end the note, attention is called to the pleiotropic characteristics of neuroendocrinology, which keeps branching off almost endlessly as bioengineering does too.