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ABSTRACT Lipids, indispensable yet structurally intricate biomolecules, serve as critical regulators of cellular function and disease progression. Conventional lipidomics, constrained by limited resolution for isomeric and low‐abundance species, has been transformed by ion mobility‐mass spectrometry (IM‐MS). This technology augments analytical power through enhanced orthogonal separation, collision cross‐section (CCS)‐based identification, and improved sensitivity. This review examines the transformative advances in IM‐MS‐driven lipidomics, focusing on three major pillars: (1) a critical evaluation of leading ion mobility spectrometry (IMS) platforms, emphasizing innovative instrument geometries and breakthroughs in resolving lipid isomers; (2) an exploration of lipid CCS databases and predictive frameworks, spotlighting computational modeling and machine learning strategies that synergize experimental data with molecular representations for high‐confidence lipid annotation; (3) emerging multi‐dimensional lipidomics workflows integrating CCS with liquid chromatography‐MS/MS to boost identification and depth, alongside mass spectrometry imaging for spatially resolved lipidomics. By unifying cutting‐edge instrumentation, computational advances, and biological insights, this review outlines a roadmap for leveraging IM‐MS to unravel lipidome complexity, catalyzing biomarker discovery and precision medicine innovation. 

Abstract Neuropeptides have tremendous potential for application in modern medicine, including utility as biomarkers and therapeutics. To overcome the inherent challenges associated with neuropeptide identification and characterization, data‐independent acquisition (DIA) is a fitting mass spectrometry (MS) method of choice to achieve sensitive and accurate analysis. It is advantageous for preliminary neuropeptidomic studies to occur in less complex organisms, with crustacean models serving as a popular choice due to their relatively simple nervous system. With spectral libraries serving as a means to interpret DIA‐MS output spectra, andCancer borealisas a model of choice for neuropeptide analysis, we performed the first spectral library mapping of crustacean neuropeptides. Leveraging pre‐existing data‐dependent acquisition (DDA) spectra, a spectral library was built using PEAKS Online. The library is comprised of 333 unique neuropeptides. The identification results obtained through the use of this spectral library were compared with those achieved through library‐free analysis of crustacean brain, pericardial organs (PO), and thoracic ganglia (TG) tissues. A statistically significant increase (Student'st‐test,Pvalue < 0.05) in the number of identifications achieved from the TG data was observed in the spectral library results. Furthermore, in each of the tissues, a distinctly different set of identifications was found in the library search compared to the library‐free search. This work highlights the necessity for the use of spectral libraries in neuropeptide analysis, illustrating the advantage of spectral libraries for interpreting DIA spectra in a reproducible manner with greater neuropeptidomic depth. 

Abstract Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well‐characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re‐evaluated. We investigate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides inCallinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms to increase almost two‐fold, from 18 to 36. Product ion‐triggered electron‐transfer/higher‐energy collision dissociation enabled the site‐specific detection of 55 intact N‐ and O‐linked glycoforms, while the faster stepped collision energy higher‐energy collisional dissociation resulted in detection of 25. Additionally, applying this workflow to five neuronal tissues enabled the characterization of 36 more glycoforms of known neuropeptides and 11 more glycoforms of nine putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro‐ and micro‐heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides. 

Crustaceans are particularly sensitive to copper toxicity, and although the downstream effects of increased copper exposure on the metabolome are often postulated and observed, they are rarely measured. To perform absolute quantification of hydrophilic small-molecule metabolites in the hemolymph of the crustacean Cancer borealis, we derivatized targeted metabolites related to copper toxicity using in-house-developed isotopic N,N-dimethyl leucine (iDiLeu) tags. Selected analytes were pooled at previously determined concentrations to serve as internal standards, and a calibration curve was generated. The sample loss was minimized by optimizing the derivatization-assisted sample cleanup using dispersive liquid–liquid microextraction (DLLME) and hydrophilic–lipophilic balancing (HLB). Calibration curves were then used for the absolute quantification of metabolites of interest following 30 min, 1 h, and 2 h exposures to 10 µM CuCl2. We found that glutamic acid was downregulated after 2 h of copper exposure, which may disrupt cellular metabolism and increase oxidative stress in crustaceans. These changes could have significant impacts on crustacean populations and the ecosystems they support. 

We establish that a complete, natural hormonal environment (hemolymph) increases the likelihood of a neuropeptide activating the gastric mill (chewing) rhythm in the crab stomatogastric ganglion (STG). The similar action of a higher neuropeptide concentration in saline, its comparable desensitizing effect to that of neuropeptide plus hemolymph on subsequent neuropeptide applications, and the absence of that neuropeptide in hemolymph suggest one or more distinct hormones act to enhance the effectiveness of the applied peptide.