Advances in venomics: Modern separation techniques and mass spectrometry

Abstract

Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components.

Keywords: Animal venoms; Drug discovery; Mass spectrometry; Separation methods; Venomic.

Fig. 1.

From venoms to drugs. A schematic representation of computational venomic approaches for discovery, identification and development of therapeutics from animal venoms [17].

Fig. 2.

Schematic representation of the ‘snake venomics’ analytical strategy developed by Calvete [34]. This strategy begins with the fractionation of the snake crude venom by RP-HPLC followed by the initial characterization of each protein fraction by a combination of N-terminal sequencing, SDS-PAGE (or 2DE), and mass spectrometric determination of the molecular masses, and eventually the cysteine (–SH and S–S) content, of the isolated components. Reproduced with permission from Ref. [134].

Fig. 3.

2-D electrophoresis workflow chart. Protein/peptides spots result from two separation steps; (i) by charge (Isoelectric focusing) and (ii) by size (SDS-PAGE).

Fig. 4.

Chromatographic profiles of Walterinnesia aegyptia venom. (A) Chromatographic profile of the whole venom was obtained using size exclusion on Sephadex G75. (B) Reversed-phase HPLC chromatogram of Walterinnesia aegyptia venom. An analytic C18 column was used. Retention time is along the x-axis. Venom components were detected at 215 nm and absorbance is indicated on the left axis. The acetonitrile gradient is shown in the HPLC graph and the percentage value corresponds to the right axis for each panel. Fraction numbers are illustrated on the top of the figure. The insert of shows the SDS-PAGE separation of the proteins collected in the fractions. Molecular markers are reported on the side of the gel [38].

Fig. 5.

Schematic representation of the three steps of OFFGEL electrophoresis principle [135]. First step, micro-wells were filled with the proteins samples diluted in a buffer. Second step, proteins start to migrate throughout the IPG gel to their isoelectric points after applying an electric field. Third step, proteins can be easily recovered in liquid phase after reaching their isoelectric points. Reproduced with permission from Ref. [135].

Fig. 6.

Venn diagram showing the number of ions detected after using SEC, RP-HPLC and OFFGEL separation techniques and those that are common between the three techniques. Reproduced with permission from Ref. [38].