Evidence-Tiered Venomics of the BotG50 Toxic Fraction from Buthus tunetanus Reveals a Diversified Neurotoxin Repertoire

Scorpion venoms represent complex pharmacological arsenals whose complete molecular inventory remains incompletely defined, particularly for North African buthid species of high medical relevance. Buthus tunetanus is among the most clinically significant scorpion species across the Maghreb. We analysed the toxic fraction BotG50, isolated by Sephadex G-50 gel-filtration, using nanoLC-HRMS/MS coupled with an evidence-tiered, five-stage bioinformatics workflow. The workflow combines Proteome Discoverer database searching, BLAST-guided homology expansion, Casanovo de novo sequencing with variant construction, signal-peptide and propeptide removal, and post-translational modification (PTM) annotation. The analysis revealed 64 components (FDR < 1%), comprising 38 multi-peptide-supported identifications (≥2 peptides) and 26 single-peptide hits, with a median sequence coverage of 33%. The dominant neurotoxic repertoire includes toxins targeting K+, Na+, and Cl- channels, α/β toxins, insect- or mammal-specific neurotoxins, and putative depressant toxins, while a minor accessory compartment comprises hyaluronidase, phospholipase A2, lipolytic peptides, and hemocyanin-like proteins. BLAST analysis revealed strong sequence homologies with peptides from Buthus israelis (32.8%), B. tunetanus (15.6%), and Mesobuthus eupeus (10.9%). Approximately 52% of final identifications (33/64 proteins) required analytical stages beyond classical FASTA database searching. This proteomic atlas provides the most detailed molecular map of BotG50 to date, constituting a critical resource for antivenom epitope design, nanobody target selection, and bioactive peptide discovery.