Comparative proteomic analysis reveals functional and evolutionary diversity in five Montivipera snake venoms

Proteomic characterization of snake venoms is essential for understanding the molecular basis of their evolution and for identifying bioactive compounds of therapeutic interest. The Montivipera species endemic to the Near and Middle East region remain poorly studied despite their interesting biological activities. Previous analyses of Montivipera venoms have provided only partial proteomic profiles, with notable discrepancies between studies. To address this gap, we conducted a proteomic analysis of five Montivipera species, including M. bornmuelleri, M. bulgardaghica, M. albizona, M. raddei and M. xanthina. We also analyzed the venom of Macrovipera lebetina ssp. to provide a broader comparative framework. These venoms were investigated using an integrated approach combining SDS-PAGE, RP-HPLC and shotgun proteomics, using both trypsin and multi-enzymatic limited digestions to maximize protein identification and coverage. SDS-PAGE and RP-HPLC analyses revealed the remarkable complexity and diversity of Montivipera venoms, which were further confirmed by shotgun proteomics, identifying between 129 and 179 proteins and peptides per species. The major protein families detected included snake venom metalloproteinases, phospholipases A₂, venom serine proteases, C-type lectins, venom vascular-endothelial growth factors, and disintegrins. Notably, the relative abundance of these protein families varied across species, suggesting interspecific differences in envenomation profiles. Comparative analysis revealed a high degree of similarity among Montivipera species, with 39 shared proteins across all five venoms. Our findings confirmed the major toxin families previously reported in Montivipera venoms and revealed the presence of several low-abundance protein families that were not previously identified. Thus, this study highlights both the conserved and unique features of Montivipera venom proteomes, offering a valuable foundation for future functional and evolutionary investigations.