Comparative peptidomics of four wasp venoms reveals extensive peptide diversity, proteolytic patterns, and predicted...

Wasp venoms possess complex compositions and diverse bioactivities, making them potential pharmacological sources. In this study, venoms from four wasp species (Vespa mandarinia, V. velutina, V. basalis, and Provespa barthelemyi) were collected by electrical stimulation and analyzed using liquid chromatography-tandem mass spectrometry. A total of 681 peptides were identified, nearly 90% of which had not been previously reported. Comparative analyses revealed pronounced species-specific signatures at both peptide and peptide family levels. Sequence-based analyses indicated that peptide release is consistent with targeted proteolytic cleavage patterns, exhibiting features resembling known substrate preferences of metalloproteases and serine proteases, rather than stochastic degradation. Bioinformatic predictions identified 291 peptides with potential bioactive properties spanning multiple functional categories, with angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP4) inhibitory activities being the most prominently represented. Among these, eight ACE inhibitory peptides and seventeen DPP4 inhibitory peptides were prioritized as candidates based on predicted safety profiles, and sequence-based analysis further identified ten putative cryptides. Overall, this study establishes the first comparative peptidomic dataset across four wasp venoms, providing insights into peptide diversity, inferred generation patterns, and predicted activities. SIGNIFICANCE: Venoms are rich sources of biologically active molecules and have historically provided templates for clinically used therapeutics. Although major protein toxins have been extensively characterized, the endogenous low-molecular-weight peptide fraction remains comparatively underexplored, particularly in social wasps, and systematic comparative resources remain limited. Venom peptidomic datasets inherently contain multiple layers of biological information, including diversification patterns, peptide origin, and potential bioactivity, yet these aspects are often interpreted independently. By integrating these dimensions, this study establishes a multi-level analytical framework for extracting biological insights and function-related information from venom peptidomes. The identification of consistent cleavage patterns suggests a degree of regulation in peptide generation, shifting the interpretation of venom peptides from degradation by-products toward biologically organized repertoires. Moreover, candidate prioritization illustrates how peptidomic datasets can generate experimentally testable functional hypotheses rather than serving solely as descriptive catalogs. The resulting dataset serves as a reference resource for cumulative comparative analyses. The analytical framework presented here also provides a transferable strategy for functional peptide discovery in other complex secretions.