Extended snake venomics by top-down in-source decay: Investigating the newly discovered Anatolian Meadow viper subspecies, Vipera anatolica senliki

Abstract

13Herein we report on the venom proteome of Vipera anatolica senliki, a recently discovered and 14 hitherto unexplored subspecies of the critically endangered Anatolian Meadow viper endemic to the 15 Antalya Province of Turkey. Integrative venomics, including venom gland transcriptomics as well as 16 complementary bottom-up and top-down proteomic analyses, were applied to fully characterize the 17 venom of V. a. senliki. Furthermore, the classical top-down venomics approach was extended to 18 elucidate the venom proteome by an alternative in-source decay (ISD) proteomics workflow using the 19 reducing matrix 1,5-diaminonaphthalene (1,5-DAN). Top-down ISD proteomics allows for disulfide 20 bond mapping as well as effective de novo identification of high molecular weight venom constituents, 21 both of which are difficult to achieve by commonly established top-down approaches. Venom gland 22 transcriptome analysis identified 42 venom transcript annotations from 13 venom toxin families. 23 Relative quantitative snake venomics revealed snake venom metalloproteinases (svMP, 42.9%) as 24 the most abundant protein family, followed by several less dominant toxin families. Online mass 25 profiling and top-down venomics provide a detailed insight into the venom proteome of V. a. senliki 26 and facilitates a comparative analysis of venom variability for the closely related subspecies, V. a. 27 anatolica.28

Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors

“…In a study on V. berus venom, the complex presence of basic phospholipases, SVSPs, LAAO, SVMP, were responsible for hemotoxicity, myotoxicity, cytotoxicity and neurotoxicity ( 63 ). V. aspis shared similar toxins with V. berus along with PLA 2 (ammodytoxin B-like PLA 2 : neurotoxic effect), SVMP inhibitor, SVMP, SVSP, and disintegrins; however, a higher presence of disintegrins were seen in V. aspis compared to V. berus ( 64 , 65 ). In separate studies on V. kaznakovi and V. anatolica venom, an abundance of SVMP was evident.…”

Mass spectrometry‐based top‐down and bottom‐up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom

“…Moreover, there are various strategies to screen scorpion venoms, from using conventional strategies for targeting one single toxin, to applying the most throughput equipment of screening for a detailed view of all toxic components. Nowadays, mass spectrometry‐based proteomic approaches are still one of the most fundamental tools to decrypt the complexity of such matrices, owing to the revolutionary advances in instrumentation and software, in addition to improvement in omics strategies (peptidomic, proteomic, transcriptomic, and genomic) [14–19]. Among the approaches that have improved significantly the proteomics workflow, there are the top‐down process, which designates a rapid analytical workflow of intact proteins, and the bottom‐up approach, which requires prior proteolytic digestion of proteins before mass spectrometry analysis.…”

Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani

“…Chromatographic conditions and ESI settings were as previously described. 65 Setup (B) LC−MS/MS experiments were done using a Vanquish ultra-high-performance liquid chromatography (UHPLC) system equipped with a 300 Å pore size, 2 mm × 150 mm column size, 3 μm particle size Supelco Discovery BIO wide C18 column thermostatted at 30 °C and hyphenated to a Q-Exactive quadrupole orbital ion trap (Thermo Fisher Scientific) as previously described. 66 MS/MS spectra were obtained in the DDA mode at a mass resolution of 140 000 (at m/z 200), and the three most abundant ions of the survey scan were selected for MS/MS.…”

“…On the other hand, in the top-down venomics (TDV) approach (Figure 1, 3a−c) front-endfractionated disulfide-bond-reduced intact polypeptide ions generated by electrospray ESI are manipulated and dissociated inside a high-resolution Fourier transform ion-trapping (e.g., orbitrap) mass spectrometer (Figure 1, 3a). 64,65,74 A benefit of TDV is that the intact mass of every proteoform is retained, overcoming the challenge of BUV regarding the characterization of small proteins that often yield an insufficient number of proteolytic peptides for unequivocal proteoform identification.…”