Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

Abstract

Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric L-amino acid oxidase (ϳ130 kDa) and the multichain toxin cobra venom factor (ϳ147 kDa). The L-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics. Molecular &

Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey

“…To circumvent these limitations, a logical solution would be the of the digestion step and the application of direct analysis of intact proteins by tandem mass spectrometry, so called top-down proteomics. Recently top-down protein analysis has been applied alone, or in combination with other venomics approaches, to study the venoms of the King cobra (Ophiophagus hannah) [21,22], the entire genus of mambas (Dendroaspis spp.) [23,24], the brown tree snake (Boiga irregularis) [6], the Okinawa habu pit viper (Protobothrops flavoviridis) [25], and several viper species from Turkey [26][27][28].…”

“…In the case of viperid species, top-down analysis typically only results in partial characterization of the venom, as a number of the main toxin components, such as high molecular weight snake venom metalloproteinases (svMPs) (>30 kDa), are challenging to efficiently ionize by denaturing electrospray ionization (ESI) and might only provide few observable fragments in tandem MS [29]. A possible way to overcome difficulties in terms of ionization of high molecular weight proteins is the application of native ESI, as described by Melani et al [22]. However native top-down mass spectrometry typically requires a special type of mass spectrometer with extended mass range and more extensive sample preparation, which makes this type of analysis more technically challenging.…”

A perspective view of top‐down proteomics in snake venom research

“…The TD MS results led to locus‐specific identification of 15 certain intact toxins in the venom of the king cobra. This venom was also investigated later by Melani et al, with the incentive of a proteoform‐centric resolution. To characterize the proteome of the venom, these authors exerted denaturing GELFrEE and IEF pre‐fractionation prior to TD MS, and identified 131 proteins and overall 184 proteoforms from 14 toxin families.…”

Drugs Containing Snake Venom: History of Development, Nomenclature, Identification

“…Яд представляет собой сложную смесь гидролитических ферментов, неэнзиматических низкои высокомолекулярных протеинов и небольшое число других органических и неорганических молекул (пигментов, минеральных компонентов и микроэлементов) [8]. Полная характеристика белков змеиного яда достаточно сложна даже при доступности современных физико-химических методов анализа из-за наличия множественных изоформ токсинов [9,10].…”

“…В настоящее время в мировой практике идентификацию змеиных ядов проводят с помощью методов иммунологического анализа [17][18][19][20], массспектрометрии [9,11,[21][22][23][24], электрофореза [10,22,25,26] или сочетания этих методов (табл. 2).…”