Imaging mass spectrometry is emerging as a powerful tool that has been applied extensively for the localization of proteins, peptides, pharmaceutical compounds, metabolites, and lipids in biological tissues. In this article, a three-dimensional mass spectral imaging (3D MSI) technique was developed to examine distribution patterns of multiple neuropeptide families and lipids in the brain of the crab Cancer borealis. Different matrix/solvent combinations were compared for preferential extraction and detection of neuropeptides and lipids. Combined with morphological information, the distribution of numerous neuropeptides throughout the 3D structure of brain was determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Different localization patterns were observed for different neuropeptide families, and isoforms displaying unique distribution patterns that were distinct from the common family distribution trends were also detected. In addition, multiple lipids were identified and mapped from brain tissue slices. any biological processes in the body involve interaction and dynamic spatial redistribution of a broad spectrum of compounds. The functions of various biological compounds in the complex tissue or organism are highly related to their locations in tissue structures. The emerging mass spectral imaging (MSI) approach provides an attractive opportunity to detect and probe the complex molecular content of tissues in an anatomical context. The majority of MSI applications have been focused on two-dimensional distribution of analytes of interest, which can only provide information for a thin section from the specimen. This technique has recently been extended to acquire images of serial sections from single specimen and provide a depth dimension to the dataset, allowing three dimensional (3D) representation of multiple analytes in the target organs [1,2]. The in-depth profiling of 3D MSI provides more comprehensive spatial information for molecules of interest, such as localization of proteins and dynamic distribution and partition of pharmaceutical compounds into various target tissues.The application of mass spectrometry to the field of neuroscience has enabled the discovery and characterization of many neuropeptides and neurohormones [3][4][5][6][7][8]. This is usually achieved by tandem MS analysis of peptides fractionated from crude nerve tissue extract. Alternatively, direct tissue methods, in which the tissue is coated with matrix and probed via matrix-assisted laser desorption/ionization (MALDI) MS analysis, enable the sensitive detection of neuropeptides in single organs [9,10] and even single cells [11,12]. More recently, the use of MSI to map the distribution of neuropeptides has gained increased attention [13][14][15]. MSI reduces the time-consuming steps of sample extraction, purification, and separation, while maintaining the topographical information about molecular distribution [16,17]. MALDI MSI enables the study of a broad mass range...
Feeding behavior is a fundamental aspect of energy homeostasis and is crucial for animal survival. This process is regulated by a multitude of neurotransmitters including neuropeptides within a complex neuroendocrine system. Given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. Here we report comparative neuropeptidomic analysis of brain and major neuroendocrine organ in a crustacean model organism in response to feeding. A multi-faceted approach employing direct tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), stable isotopic labeling of neuropeptide extracts for quantitation, and mass spectrometric imaging (MSI) has been employed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and the pericardial organ (PO) in the crab Cancer borealis. Multiple neuropeptides exhibited changes in abundance after feeding, including RFamides, Cancer borealis tachykinin related peptides (CabTRPs), RYamides, and pyrokinins. By combining quantitative analysis of neuropeptide changes via isotopic labeling of brain extract and MSI mapping of neuropeptides of brain slices, we identified the boundary of olfactory lobe (ON) and median protocerebrum (MPC) area as two potential feeding centers in the crab brain.
The lobster Homarus americanus has long served as an important animal model for electrophysiological and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biological functions. Using nanoLC ESI Q-TOF MS/MS and three types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/extract analysis and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resolution and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide analysis in terms of peptidome coverage, sensitivity, mass spectral resolution and capability for de novo sequencing.
Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C 18 micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were detected, including both known ones and new peptides whose functions remain to be characterized. (J Am Soc Mass Spectrom 2009, 20, 708 -718)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.