It is time for top-down venomics

Abstract

The protein composition of animal venoms is usually determined by peptide-centric proteomics approaches (bottom-up proteomics). However, this technique cannot, in most cases, distinguish among toxin proteoforms, herein called toxiforms, because of the protein inference problem. Top-down proteomics (TDP) analyzes intact proteins without digestion and provides high quality data to identify and characterize toxiforms. Denaturing top-down proteomics is the most disseminated subarea of TDP, which performs qualitative and quantitative analyzes of proteoforms up to ~30 kDa in high-throughput and automated fashion. On the other hand, native top-down proteomics provides access to information on large proteins (> 50 kDA) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry. The use of native and denaturing top-down venomics introduced novel and useful techniques to toxinology, allowing an unprecedented characterization of venom proteins and protein complexes at the toxiform level. The collected data contribute to a deep understanding of venom natural history, open new possibilities to study the toxin evolution, and help in the development of better biotherapeutics.

Venomics: A Mini-Review

“…Top-down proteomics is more attractive in theory as it studies the proteins as intact entities, and has the advantage of simultaneously measuring all modifications that occur on the same molecule and enabling identification of the precise proteoform. However, because each protein may have multiple proteoforms (toxiforms in venom [ 50 ]) that may have different functions, top-down proteomics is experimentally and computationally more challenging [ 49 ]. Additionally, top-down proteomics still faces challenges imposed by current limitations on the front-end fractionation of complex mixtures and instrument-related limitations, particularly in relation to high mass proteins [ 51 ].…”

“…The availability of relevant protein or nucleic acid databases is frequently a limitation in proteomic studies [ 15 ]. In general, the traditional bottom-up approaches have the disadvantage of typically failing to provide complete protein sequence coverage and preventing the distinction between different related protein species, particularly proteoforms and protein isoforms, and is known as the protein inference problem [ 50 , 52 ].…”

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

“…Ultimately, employing top-down proteomics combined with a front-end chromatographic separation is a suitable alternative to uncover existing proteoforms, PTMs and to identify toxin components by tandem MS or disulfide bond mapping. 11, 61…”

Current research into snake antivenoms, their mechanisms of action and applications

“…With the development of improved pharmacological techniques, the functional aspects of different toxin groups relevant to envenoming syndromes in humans have been better understood [37][38][39][40][41]. In addition, proteomic and transcriptomic approaches have been used to investigate venom composition and variation (venomics), which has revolutionized our understanding of snake venom [42][43][44][45][46][47].…”