Biology subjects

Ibarra, A. E.

Publications and source records attributed to Ibarra, A. E..

4 recordsLinked to original sources

bioRxiv2026

12-plex DiLeu enables robust quantification of the feeding neuropeptidome

Understanding the feeding-induced neuropeptidome cascade requires analytical strategies capable of quantifying low-abundance, highly modified peptides across multiple tissues and time points. Herein, we apply 12-plex N,N-dimethyl leucine (DiLeu) isobaric labeling to perform the first multiplexed, tissue-wide, temporal quantitation of the Cancer borealis feeding neuropeptidome. This approach enabled sensitive measurement of neuropeptides across five tissues over six timepoints, revealing distinct regulatory patterns. The pericardial organ (PO) showed rapid early upregulation followed by suppression aligned with foregut emptying, whereas the thoracic ganglion (TG) displayed inverse and strongly condition-dependent responses, indicating previously unrecognized neuromodulatory roles. Single-residue variants and post-translational modifications, including pyro-Glu formation and amidation, produced markedly different temporal profiles, underscoring the functional specificity of closely related isoforms. We further identify differential regulation of proctolin and its amidated form, suggesting modified variants may contribute uniquely to feeding physiology. Collectively, these results establish multiplexed DiLeu labeling as a powerful platform for quantitative neuropeptidomics and reveal new dimensions of peptide-mediated feeding regulation.

neuroscience↗

bioRxiv2026

Bridging genomes and peptidomes: hybrid sequencing reveals conserved bioactive peptides in crustaceans

Endogenous peptides are critical regulators of signaling and immunity but remain difficult to characterize in organisms with incomplete genomic annotation. We developed a hybrid discovery platform that integrates transformer-based de novo sequencing (Casanovo), neuropeptide-focused database searching (EndoGenius), and empirical false discovery rate estimation via NovoBoard. This pipeline enables confident identification of endogenous peptides while expanding coverage beyond conventional database-only or de novo-only approaches. Applied to neuroendocrine tissues from Callinectes sapidus and Cancer borealis, the workflow revealed numerous high-abundance novel peptides and provided structural and genomic support for their biological relevance. Notably, we report the first histone-2A-derived antimicrobial peptide in the C. sapidus and characterize naturally occurring sequence variants. We also identified unexpected peptide homologies between crustaceans and Rattus norvegicus, enabling annotation of conserved housekeeping proteins in sparsely annotated genomes. This hybrid platform establishes a scalable, open-source strategy for advancing neuropeptidomics and endogenous peptide discovery in emerging model organisms.

bioinformatics↗

bioRxiv2025

HILIC-Enabled Mass Spectrometric Discovery of Novel Endogenous and Glycosylated Neuropeptides in the American Lobster Nervous System

Neuropeptides are a highly conserved and diverse class of intercellular signaling molecules that regulate a broad range of neural and hormonal processes across animal phyla. The American lobster, Homarus americanus, has long served as a powerful invertebrate model for the discovery and functional investigation of neuropeptides. Among common post-translational modifications (PTMs) found in neuropeptides, glycosylation remains underexplored due to the inherently low in vivo abundance and intrinsically complex structural heterogeneity. In this study, we employed hydrophilic interaction liquid chromatography (HILIC) enrichment coupled with oxonium-ion triggered EThcD fragmentation strategy to simultaneously profile novel endogenous and glycosylated neuropeptides across eight distinct neural tissues and neuroendocrine organs of Homarus americanus. This integrative mass spectrometry-based approach led to the identification of 154 endogenous neuropeptides derived from 25 families, approximately one-third of which are newly reported, and uncovered 24 glycosylated neuropeptides in this species for the first time. These peptides exhibit strong tissue-specific expression, distinct proteolytic cleavage patterns, and confidently localized glycosylation sites. Our results highlight the utility of integrated sampling enrichment and hybrid fragmentation strategies for deep neuropeptidomic profiling and provide a valuable resource for future studies on the functional roles of newly identified neuropeptides and glycosylation in crustacean neuromodulation and peptidergic signaling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/661634v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@9a1745org.highwire.dtl.DTLVardef@6decc4org.highwire.dtl.DTLVardef@d34256org.highwire.dtl.DTLVardef@1a83b4c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Unlocking the Neuropeptidome using a Novel Endogenous Peptidomics Framework

Endogenous peptides have garnered increasing attention over the past decade driven by the development of advanced analytical methods. However, large-scale investigations of peptides as potential disease biomarkers or drug candidates are still hindered by their challenging biochemical properties and the scarcity of specialized analytical tools. Among these, neuropeptides are particularly challenging to study due to their low in vivo concentration, rapid turnover rate, and high structural variability. Data-independent acquisition (DIA) mass spectrometry (MS) has shown great ability in profiling low-abundance ions. Nevertheless, most available DIA analytical tools are designed for proteomics studies and are not suitable for endogenous peptides, as there is no set enzymatic cleavage for these peptides. Here, we introduce the novel EndoGenius platform, paired with DIA-NN, to achieve high-confidence neuropeptide identification using an updated spectral library for DIA MS analysis. By employing orthogonal offline fractionation, ion mobility instrumentation, and an optimized database searching algorithm specifically for neuropeptides, we have constructed the largest crustacean neuropeptide spectral library to date. With this library, in combination with neural networking technology, we report a 100-fold increase in the number of neuropeptides identified in all Cancer borealis tissues analyzed. We also cross-validated these findings with transcriptomics data to enhance identification confidence. This workflow presents a novel analytical framework for DIA peptidomics analysis, offering a robust approach to studying neuropeptides and other endogenous peptides. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/659356v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1bae342org.highwire.dtl.DTLVardef@9e26d8org.highwire.dtl.DTLVardef@1084f06org.highwire.dtl.DTLVardef@7c1371_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisWe present a framework that capitalizes on robust analytical innovations and an optimized bioinformatics pipeline to provide the most comprehensive snapshot of the crustacean neuropeptidome to-date.

neuroscience↗