Snakebite envenoming constitutes a significant global health issue, particularly in Africa, where venomous species such as Echis vipers and Dendroaspis mambas pose substantial risks to human health. This study employs a standardized venomics workflow to comprehensively characterize and comparatively quantify the venom composition of nine medically relevant snake species chosen from among the deadliest in Africa. Utilizing shotgun venom proteomics and venom gland transcriptomics, we report detailed profiles of venom complexity, highlighting the relative abundance of dominant toxin families such as three-finger toxins and Kunitz-type proteins in Dendroaspis, and metalloproteinases and phospholipases A₂ in Echis. We delineate here the relative abundance and structural diversity of venom components. Key to our proteomic approach is the implementation of Multi-Enzymatic Limited Digestion (MELD), which improved protein sequence coverage and enabled the identification of rare toxin families such as hyaluronidases and renin-like proteases, by multiplying the overlap of generated peptides and enhancing the characterization of both toxin and non-toxin components within the venoms. The culmination of these efforts resulted in the construction of a detailed toxin database, providing insights into the biological roles and potential therapeutic targets of venom proteins and peptides. The findings here compellingly validate the MELD technique, reinforcing its reproducibility as a valuable characterization approach applied to venomics. This research significantly advances our understanding of venom complexity in African snake species, including representatives of both Viperidae and Elapidae families. By elucidating venom composition and toxin profiles, our study paves the way for the development of targeted therapies aimed at mitigating the morbidity and mortality associated with snakebite envenoming globally.

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.

Scorpionism is a growing public health concern in Brazil, with the Amazon region presenting the highest mortality rates but remaining understudied, especially regarding local scorpion venoms composition. This study presents the first comprehensive biochemical characterization of venoms from three Amazonian species—Tityus metuendus (TmetuV), Tityus silvestris (TsilvV), and Brotheas amazonicus (BamazV)—using an integrated approach combining Multi-Enzymatic Limited Digestion (MELD)-based bottom-up proteomics, high-resolution LC-MS/MS, chromatography, zymography, and enzymatic assays. Tityus serrulatus venom was included as a reference. Significant biochemical differences were observed: TsilvV was rich in 20–30 kDa proteins and showed strong metalloprotease activity; BamazV exhibited high molecular weight proteins and potent phospholipase A₂ (PLA₂) activity but lacked proteolytic and fibrinogenolytic activities; TmetuV showed the highest hyaluronidase activity and abundance of α-KTx neurotoxins. Zymography revealed a conserved ~45 kDa hyaluronidase in all species. Three novel components were partially characterized: BamazPLA₂ (Group III PLA₂), Tmetu1 (37-residue α-KTx), and TsilvMP_A (a metalloprotease homologous to antarease). This is the first application of MELD-based proteomics to Amazonian scorpion venoms, revealing molecular diversity and functional divergence within Tityus and Brotheas, emphasizing the need for region-specific antivenoms. These findings provide a foundation for future pharmacological studies and the discovery of bioactive peptides with therapeutic potential.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom—polyclonal antibodies isolated from the plasma of hyperimmunised animals—which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebite and 3-fold more victims suffer life-altering disabilities. Current treatment of snakebite relies solely on antivenom – polyclonal antibodies isolated from the plasma of hyperimmunised animals – which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein with 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology, mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.