Exploring Diversity of Conopeptides and Revealing Novel Conoinsulins from <i>Conus betulinus</i> by Proteomic Analyses

The venom of cone snails, a potent weapon for predation and defense, contains diverse bioactive peptides (known as conopeptides, or conotoxins) that target various ion channels and receptors, offering potential as pharmacological tools or therapeutics. While transcriptomic studies have expanded conopeptide databases, proteomic validation remains limited. Here, we integrated two high-resolution mass spectrometry platforms to explore conopeptide diversity in <i>Conus betulinus</i>. A total of 283 conopeptides were identified, with 268 classifiable into known gene superfamilies or homology classes, while 15 unclassified conopeptides represent novel superfamilies. There were 46 newly discovered sequences and five new cysteine frameworks. Notably, we report the first proteomic identification of two novel conoinsulins in <i>C. betulinus</i>, Con-ins Be1 and Con-ins Be2. Both of them were predicted to retain insulin's canonical A/B-chain architecture. Structure modeling using the AlphaFold2 multimer suggested that Con-ins Be1 has a four-disulfide-bond arrangement, differing from the three disulfide bonds found in vertebrate insulin. In contrast, Con-ins Be2 was predicted to have three disulfide bonds, consistent with the structure of the vertebrate insulin. In summary, our study not only expanded the conopeptide repository but also provided two novel conoinsulins that may serve as pharmacological tools for insulin system research and merit further investigation.