No abstract

Neuropeptides are important mediators in the functionality of the brain and other neurological organs. Because neuropeptides exist in a wide range of concentrations, appropriate characterization methods are needed to provide dynamic, chemical, and spatial information. Mass spectrometry and compatible tools have been a popular choice in analyzing neuropeptides. There have been several advances and challenges, both of which are the focus of this review. Discussions range from sample collection to bioinformatic tools, although avenues such as quantitation and imaging are included. Further development of the presented methods for neuropeptidomic mass spectrometric analysis is inevitable, which will lead to a further understanding of the complex interplay of neuropeptides and other signaling molecules in the nervous system.

Environmental fluctuations, such as salinity, impose serious challenges to marine animal survival. Neuropeptides, signaling molecules involved in the regulation process, and the dynamic changes of their full complement in the stress response have yet to be investigated. Here, a MALDI-MS-based stable isotope labeling quantitation strategy was used to investigate the relationship between neuropeptide expression and adaptability of Carcinus maenas to various salinity levels, including high (60 p.p.t.) and low (0 p.p.t.) salinity, in both the crustacean pericardial organ (PO) and brain. Moreover, a high salinity stress time course study was conducted. MS imaging (MSI) of neuropeptide localization in Carcinus maenas PO was also performed. As a result of salinity stress, multiple neuropeptide families exhibited changes in their relative abundances, including RFamides (e.g. APQGNFLRFamide), RYamides (e.g. SSFRVGGSRYamide), B type-allatostatins (AST-B) (e.g. VPNDWAHFRGSWamide), and orcokinins (e.g. NFDEIDRSSFGFV). The MSI data revealed distribution differences in several neuropeptides (e.g. SGFYANRYamide) between color morphs, but salinity stress appeared to not have a major effect on the localization of the neuropeptides.

Temperature changes influence the reaction rates of all biological processes, which can pose dramatic challenges to cold-blooded organisms, and the capability to adapt to temperature fluctuations is crucial for the survival of these animals. In order to understand the roles that neuropeptides play in the temperature stress response, we employed a mass spectrometry-based approach to investigate the neuropeptide changes associated with acute temperature elevation in three neural tissues from the Jonah crab Cancer borealis. At high temperature, members from two neuropeptide families, including RFamide and RYamide, were observed to be significantly reduced in one of the neuroendocrine structures, the pericardial organ, while several orcokinin peptides were detected to be decreased in another major neuroendocrine organ, the sinus gland. These results implicate that the observed neuropeptides may be involved with temperature perturbation response via hormonal regulation. Furthermore, a temperature stress marker peptide with the primary sequence of SFRRMGGKAQ (m/z 1137.7) was detected and de novo sequenced in the circulating fluid (hemolymph) from animals under thermal perturbation.

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography–tandem mass spectrometry (nanoRPLC–MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.