HILIC-MS Profiling Reveals Novel Endogenous and Glycosylated Neuropeptides in the American Lobster Nervous System

The American lobster (Homarus americanus) has long served as a powerful invertebrate model for researcher’s 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. Research conducted at the University of Wisconsin-Madison employed hydrophilic interaction liquid chromatography (HILIC) enrichment coupled with oxonium-ion triggered electron-transfer/higher-energy collisiondissociation(EThcD) fragmentation to simultaneously profile novel endogenous and glycosylated neuropeptides across eight distinct neural tissues and neuroendocrine organs of lobsters. A paper based on their study was published in bioRxiv (1).

Initially encoded in the genome as part of larger precursor proteins known as preprohormones, mature bioactive neuropeptides are released following multiple proteolytic cleavages and extensive post-translational modifications (PTMs), exhibiting high sequence diversity, structural similarity, and numerous PTMs. One of the common PTMs found in neuropeptides is glycosylation, where a glycan (a group of monosaccharides) is attached to the side chain of the peptide backbone, capable of altering bioactivity, receptor binding, and peptide stability (2–4)

The American lobster has long served as valuable model organisms in neuroscience research, due to their relatively simple, accessible, and well-defined neural circuits. These circuits express a wide array of neuropeptides that are structurally and functionally homologous to those found in vertebrates (5). The nervous system of the lobster is made up of multiple anatomically distinct yet functionally interconnected regions, including both neural tissues and neuroendocrine organs. These regions engage in reciprocal signaling, where neuropeptides act both locally within specific circuits and systemically as hormonal messengers to coordinate neuromodulation, neuronal plasticity, and physiological responses (6)

Lobsters for this study were purchased from Global Market and Food Hall (Madison, WisconsinI) and allowed to acclimate to artificial seawater tanks (made with Instant Ocean Sea Salt, 10– 12°C, alternating 12-hr light/dark cycle) for at least two weeks before the study began. The lobsters were housed, treated, and sacrificed in accordance with the animal care and study protocol approved by the UW-Madison Animal Care and Use Committee. Lobsters were cold anesthetized on ice for 30 min prior to the dissection conducted in chilled physiological saline solution. Eight neural tissues, including a pair of sinus glands (SG), the brain, the oesophageal ganglion (OG), a pair of commissural ganglia (CoG), the stomatogastric ganglion (STG), the thoracic ganglion (TG), a pair of pericardial organs (PO), and the cardiac ganglion (CG), were collected and immediately placed in 200 µL of an ice-cold acidified methanol mixture (90% methanol, 9% water, and 1% glacial acetic acid), snap-frozen on dry ice, and stored at -80°C until needed.

Due to the limited amount of the endogenous glycosylated neuropeptide per tissue, samples were pooled from 10 to 20 lobsters to obtain 100–300 µg of starting material per analysis. All samples were dried and stored at -80°C for eventual HILIC enrichment, incorporated to clean-up and selectively enrich glycosylated and endogenous neuropeptides. Because HILIC involved multiple wash steps that result in significant sample loss, a relatively large amount of starting material was required. Lobster tissues were pooled according to their relative size and neuropeptide content, with 10 lobsters pooled per sample for brain, TG, CG, paired CoGs, SGs, POs, and 20 lobsters for smaller STG and OG tissues, yielding 100–300 µg material per tissue. Unlike tryptic peptides, neuropeptides vary in length ranging from 4 to more than 80 amino acids and contain a higher proportion of basic residues, requiring conventional HILIC protocols for tryptic glycopeptides to be optimized. Increasing the organic composition reconstitution and wash buffers from 80% to 95% significantly improved neuropeptide retention on HILIC materials (7). Using this optimized workflow, HILIC enrichment substantially improved neuropeptide recovery, yielding 20% to 50% more neuropeptides than non-enriched samples across all eight neural tissues (1).

The researchers reported that through the incorporation of HILIC enrichment into their sample preparation workflow, they significantly expanded its known neuropeptidome landscape, identifying 154 unique neuropeptides from 25 families, with approximately one-third of them being reported for the first time. Also, by using oxonium ion-triggered EThcD fragmentation approach, they characterized 28 O-linked glycosylated neuropeptides for the first time, greatly advancing the understanding of the American lobster nervous system, including the elucidation of key cleavage patterns, peptide properties, tissue-specific, and species-specific distribution that further suggests their distinct roles in neuromodulation and neuroendocrine regulation. These results, they believe, highlight the utility of integrated sampling enrichment and hybrid fragmentation strategies for deep neuropeptidomic profiling, providing an important resource for future studies on the functional roles of newly identified neuropeptides and glycosylation in crustacean neuromodulation and peptidergic signaling (1).

References

1. Tran, V. N. H.; Lu, G.; Duong, T. et al. HILIC-Enabled Mass Spectrometric Discovery of Novel Endogenous and Glycosylated Neuropeptides in the American Lobster Nervous System. bioRxiv 2025, 2025 (6), 661634. DOI: 10.1101/2025.06.26.661634
2. Apostol, C. R.; Hay, M.; Polt, R. Glycopeptide Drugs: A Pharmacological Dimension Between “Small Molecules” and “Biologics.” Peptides 2020, 131, 170369. DOI: 10.1016/j.peptides.2020.170369
3. Madsen, T. D.; Hansen, L. H.; Hintze, J. et al. An Atlas of O-Linked Glycosylation on Peptide Hormones Reveals Diverse Biological Roles. Nat. Commun. 2020, 11 (1), 4033. DOI: 10.1038/s41467-020-17473-1
4. Moradi, S. V.; Hussein, W. M.; Varamini, P. et al. Glycosylation, An Effective Synthetic Strategy to Improve the Bioavailability of Therapeutic Peptides. Chem. Sci. 2016, 7 (4), 2492- 2500. DOI: 10.1039/c5sc04392a
5. Fields, L.; Dang, T. C.; Tran, V. N. H. et al. Decoding Neuropeptide Complexity: Advancing Neurobiological Insights from Invertebrates to Vertebrates through Evolutionary Perspectives. ACS Chem. Neurosci. 2025,16 (9), 1662-1679. DOI: 10.1021/acschemneuro.5c00053
6. Marder, E.; Bucher, D. Understanding Circuit Dynamics Using the Stomatogastric Nervous System of Lobsters and Crabs. Annu. Rev. Physiol.2007, 69, 291-316. DOI: 10.1146/annurev.physiol.69.031905.161516
7. Phetsanthad, A.; Roycroft, C.; Li, L. Enrichment and Fragmentation Approaches for Enhanced Detection and Characterization of Endogenous Glycosylated Neuropeptides. Proteomics 2023, 23 (3-4), 2100375. DOI: 10.1002/pmic.202100375