Sexual Dimorphism and Intraspecific Toxin Variation in Crotalus durissus terrificus Venom: Insights from Proteomics, Peptidomics, and Structural Modeling

The venom of the South American rattlesnake Crotalus durissus terrificus (Cdt) is renowned for its minimalist yet highly toxic arsenal. Intraspecific variability in expressed proteoforms and toxin content has been previously observed in Cdt venom, resulting in heterogeneous biological activities. In light of this, our study conducted an in-depth mass spectrometry-based analysis, combined with structural prediction to comprehensively assess Cdt venom variability and sexual dimorphism. Using peptidomic analysis, we identified 76 native peptides within Cdt venom, predominantly (63%) associated with noncanonical processing of crotoxin subunits, and 9 novel peptides via de novo sequencing. Quantitative analysis showed increased abundance of two crotapotin-derived peptides in females, while three novel peptides were significantly increased in males. Through quantitative proteomics, we characterized a repertoire of 33 proteins, with over one-third displaying sex-based differences in abundance. Furthermore, variant analysis revealed 12 novel toxin variants, generally at lower abundance than previously described proteoforms. Predictive three-dimensional structure modeling revealed conserved scaffolds, suggesting preservation of their biological activities. Addressing venom variability provides valuable insights into the intricate variability of Cdt venom, contributing to the understanding of its biological, medical, and biotechnological implications.