Mass Spectrometry of Peptides in Neuroscience (original) (raw)
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Peptides in the Brain: Mass Spectrometry–Based Measurement Approaches and Challenges
Annual Review of Analytical Chemistry, 2008
The function and activity of almost every circuit in the human brain are modified by the signaling peptides (SPs) surrounding the neurons. As the complement of peptides can vary even in adjacent neurons and their physiological actions can occur over a broad range of concentrations, the required figures of merit for techniques to characterize SPs are surprisingly stringent. In this review, we describe the formation and catabolism of SPs and highlight a range of mass spectrometric techniques used to characterize SPs. Approaches that supply high chemical information content, direct tissue profiling, spatially resolved data, and temporal information on peptide release are also described. Because of advances in measurement technologies, our knowledge of SPs has greatly increased over the last decade, and SP discoveries will continue as the capabilities of modern measurement approaches improve.
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.
Direct Sequencing of Neuropeptides in Biological Tissue by MALDI−PSD Mass Spectrometry
Analytical Chemistry, 1999
Dissected tissue pieces of the pituitary pars intermedia from the amphibian Xenopus laevis was directly subjected to matrix-assisted laser desorption/ionization (MALDI) mass analysis. The obtained MALDI peptide profile revealed both previously known and unexpected processing products of the proopiomelanocortin gene. Mass spectrometric peptide sequencing of a few of these neuropeptides was performed by employing MALDI combined with postsource decay (PSD) fragment ion mass analysis. The potential of MALDI-PSD for sequence analysis of peptides directly from unfractionated tissue samples was examined for the first time for the known desacetyl-r-MSH-NH 2 and the presumed vasotocin neuropeptide. In addition, the sequence of an unknown peptide which was present in the pars intermedia tissue sample at mass 1392.7 u was determined. The MALDI-PSD mass spectrum of precursor ion 1392.7 u contained sufficient structural information to uniquely identify the sequence by searching protein sequence databases. The determined amino acid sequence corresponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ("vasotocinyl-GKR"), indicating incomplete processing of the vasotocin precursor protein in the pituitary pars intermediate of X. laevis. Both vasotocin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of these two peptides reveals three different forms of characteristic fragment ions of the cysteine side chain: peptide-CH 2 -SH (regular mass of Cys-containing fragment ions), peptide-CH 2 -S-SH (regular mass + 32 u) and peptidedCH 2 (regular mass -34 u) due to cleavage on either side of the sulfur atoms.
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...
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...
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.
Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry
Journal of Proteomics, 2012
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.
Peptide identifications and false discovery rates using different mass spectrometry platforms
Talanta, 2018
Characterization of endogenous neuropeptides produced from post-translational proteolytic processing of precursor proteins is a demanding task. A variety of complex prohormone processing steps generate molecular diversity from neuropeptide prohormones, making in silico neuropeptide discovery difficult. In addition, the wide range of endogenous peptide concentrations as well as significant peptide complexity further challenge the structural characterization of neuropeptides. Liquid chromatography-mass spectrometry (MS), performed in conjunction with bioinformatics, allows for high-throughput characterization of peptides. Mass analyzers and molecular dissociation techniques render specific characteristics to the acquired data and thus, influence the analysis of the MS data using bioinformatic algorithms for follow-up peptide identification. Here we evaluated the efficacy of several distinct peptidomic workflows using two mass spectrometers, the Thermo Orbitrap Fusion Tribrid and Bruke...