ABSTRACT: We report the venom proteome of Vipera anatolica senliki, a newly discovered subspecies of the Anatolian Meadow viper endemic to the Antalya Province in Turkey. Integrative venomics, including venom gland transcriptomics as well as complementary bottom-up and top-down proteomic analyses, were applied to fully characterize the venom of V. a. senliki. Additionally, we extend the top-down venomics approach to elucidate the venom proteome by an in-source decay-driven (ISD) workflow using the reducing matrix 1,5-diaminonaphthalene (DAN). Our venomic in-source decay protocol allowed disulfide bond mapping as well as an effective de novo identification of high molecular weight venom constituents, both of which are difficult to achieve by established top-down approaches. Venom gland transcriptome analysis identified 42 venom genes annotations relating to number 13 venom toxin families. Relative quantitative snake venomics revealed snake venom metalloproteinases (svMP, 42.9%) as most abundant protein family, followed by less abundant toxin families as cysteine-rich secretory proteins (CRISP, 9.9%), phospholipases A2 (PLA2, 8.2%), snake venom serine proteinases (svSP, 7.2%) and C-type lectin-like proteins(CTL, 4.6%) as well as disintegrins (DI, 1.9%), Kunitz-type serine protease inhibitor (KUN, 1.2%), L-amino acid oxidase (LAAO, 0.1%) and non-annotated (n/a, 0.5%) were identified. Furthermore, a high content of diverse peptides (23.5%), e.g. svMP-inhibitor (svMP-i, 5.9%) and bradykinin potentiating peptides (BPP; 0.6%) were found. Top-down venomics showed the presence of DI, KUN, and several PLA2 proteoforms that were also previously reported in the closely related subspecies V. anatolica anatolica.
Benjamin-Florian Hempel 
