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.

Keywords: Denaturing top-down proteomics; Native top-down proteomics; Top-down proteomics; Toxiforms; Venomics.

Fig. 1

Graphical representation of generic bottom-up (left panel) and top-down (right panel) venomics experiments. On the left panel, venom proteins are reduced, alkylated, enzymatically digested, and submitted to LC-MS/MS for peptide identification; toxins are inferred resulting in more protein possibilities than the original number of toxiforms (inference problem). On the right panel, venom is pre-fractionated before LC-MS/MS resulting in the identification and characterization of all toxiforms present in the initial sample

Fig. 2

Fragmentation maps of acidic phospholipase A₂ 2 (Q9DF33) and weak toxin DE-1 (P01412) toxiforms from Ophiophagus hannah venom. Gray squares represent amino acid alterations from the deposited sequence in UniProt database and the orange square represents a pyroglutamic acid. Data from Melani et al. [13] used to create this image are freely available at ProteomeExchange identifier PXD003403. Fragmentation maps, scoring and residue coverage were obtained using the software ProSight Lite