Mass Spectrometry-Based Molecular Profiling of Snake Venom System: Proteomics, Lipidomics and Mass Spectrometry Imaging

Abstract

Animal venoms are rich natural sources of molecules with a broad spectrum of biological and pharmacological activities. Their biochemical and toxic properties have fascinated humankind for epochs. While these toxins have primarily evolved to facilitate prey capture and defense against enemies, they have potential value for translation into human therapeutics. Ten FDA-approved toxin-based medications are currently marketed according to the specificity and potency for particular molecular targets. Continuous improvement and development of bioanalytical techniques, particularly mass spectrometry, enable accurate qualitative and quantitative determination and even spatial localization of biomolecules in various biological systems. These technological advances have significantly increased our knowledge of the structure and function of venom components and their biological interactions, recognizing their potential benefits, e.g., novel biomedical and diagnostic tools. Here, we performed different studies related to the medically important saw-scaled or carpet vipers (genus Echis), which are considered to cause higher global snakebite mortality than any other snake. Among all species of Echis genus, Echis carinatus sochureki (ECS) is a widely distributed snake species. The species is also found across the thirteen provinces of Iran, where it is assumed to be responsible for most snakebite envenomings. We collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to understand the viper life better. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD50 (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly similar venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families, including the most abundant snake venom metalloproteinases (SVMPs, 29−34%); phospholipase A2 (PLA2s, 26−31%); snake venom serine proteinases (SVSPs, 11−12%); l-amino acid oxidases (LAOs, 8−11%), c-type lectins/lectin-like (CTLs, 7−9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies. Moreover, we employed autofocusing atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to investigate endogenous biomolecular localizations and distribution patterns in the venom glands of ECS. For this reason, fresh-freezing and formalin-fixating sample preparations were tested for the gland to obtain data from the morphologically "intact state" of the gland sections. Subsequently, MSI was conducted with 12 μm pixel resolution for both types of preparations, and the lateral distributions of the metabolites were identified. Experiments revealed that lipids belonging to the classes of PC (phosphatidylcholines), SM (sphingomyelins), PE (phosphatidylethanolamines), PS (phosphatidylserines), PA (phosphatidic acids), and TG (triglycerides) are present in the venom gland. PC (32:0) and SM (36:1) were found to be specifically located in the areas where cells are present. The snake venom metalloprotease inhibitor pEKW (m/z 444.2233) was identified in the venom by top-down LC−MS/MS and localized by AP-SMALDI MSI in the gland across secretory epithelial cells. The peptide can inhibit the venom’s enzymatic activity during long-term storage within the venom gland and thus protect its tissue. Finally, with a high degree of spectral similarities, we concluded that formalin-fixed tissue, in addition to its high ability to preserve tissue morphology, can be considered as an alternative method to fresh-frozen tissue in the case of lipid and peptide MS imaging in venom gland tissues.