Proteomic and Transcriptomic Techniques to Decipher the Molecular Evolution of Venoms

Abstract

Nature's library of venoms is a vast and untapped resource that has the potential of becoming the source of a wide variety of new drugs and therapeutics. The discovery of these valuable molecules, hidden in diverse collections of different venoms, requires highly specific genetic and proteomic sequencing techniques. These have been used to sequence a variety of venom glands from species ranging from snakes to scorpions, and some marine species. In addition to identifying toxin sequences, these techniques have paved the way for identifying various novel evolutionary links between species that were previously thought to be unrelated. Furthermore, proteomics-based techniques have allowed researchers to discover how specific toxins have evolved within related species, and in the context of environmental pressures. These techniques allow groups to discover novel proteins, identify mutations of interest, and discover new ways to modify toxins for biomimetic purposes and for the development of new therapeutics.

Keywords: high-performance liquid chromatography; mass spectrometry; predator; prey; toxin; venom.

Figure 1

Classic workflow for studying toxin components from venoms. In most studies, the series of experiments employed to elucidate the molecular components of venoms involve studying the venom gland and the venom itself separately. The venom gland is subject to RNA extraction, mRNA enrichment, cDNA library preparation and RNA sequencing to obtain the gland’s transcriptome. In parallel, the whole venom is fractionated using any number of separation techniques, high-performance liquid chromatography (HPLC) is pictured above. These fractions are subject to mass spectrometry to identify peptides and obtain a proteome with the gland’s transcriptome (obtained from RNA sequencing) being utilized as a reference database for the mass spectrometry.

Figure 2

Top-down vs. Bottom-up proteomics approaches. The two main approaches in discerning toxins from one another in venoms are top-down and bottom-up proteomics. Top-down proteomics describes an approach whereby a venom’s whole proteins (notice: native, folded proteins in tubes on top left) are holistically analyzed without the need for breaking proteins down into their constituent fragments. On the other hand, bottom-up proteomics refers to techniques that involve the denaturing of whole proteins (notice: unfolded proteins on top right) into fractions, and the study of these protein fractions separately, before reassembling these fragments into proteins in silico to identify a venom’s constituent toxin proteins. Typically, top-down proteomics is more accurate in discerning between closely related toxins with minimal sequence variation or toxins with post-translational modifications.

Figure 3

Overview of most commonly used sample separation techniques in venomics. (A) A molecular weight sieve is used for size exclusion chromatography to separate samples based on their size or molecular weight. (B) High-performance liquid chromatography (HPLC) is used to separate analytes in a sample based on their polarity. Analytes interact with a column (stationary phase) differently based on their polarity, causing them to elute at different speeds. The eluted molecules are detected by either a spectroscopic or an electrochemical detector, with a readout available to the experimenter describing the eluate’s absorbance at different time points. (C) Capillary isoelectric focusing (CIEF) is a separation technique that separates molecules based on their isoelectric points in fused silica capillary tubes. (D) The Agilent 2100 Bioanalyzer is an innovative new “venom-on-a-chip” technology that incorporates a variation of CIEF in which microchannels host an electrophoretic separation of proteins, which are then detected via fluorescence. The software then transforms this data into gel-like images and electropherograms for easy interpretation.

Figure 4

Ionization techniques for mass spectrometry. (A) Matrix-assisted laser desorption/ionization (MALDI) is an ionization technique used for mass spectrometry that involves sublimating an analyte molecule embedded in a matrix of small molecules. During this sublimation, the analyte itself is not fragmented, but rather ionized thanks to the protonating/deprotonating properties of the matrix molecules. (B) Electrospray ionization (ESI) is another ionization technique that consists of the use of high voltage and a nebulizing gas to vaporize a solvent, containing analytes, into an aerosol. The remaining solvent in these droplets is evaporated, leaving a charged droplet, which undergoes Coulomb fission resulting in charged progeny droplets. From these droplets, naked charged analytes are detected by the device’s detector to investigate their m/z ratio.

Figure 5

Detection techniques for mass spectrometry. (A) Time-of-flight (TOF) is the most common detection technique in mass spectrometry and is often coupled with MALDI. Ionized analyte particles travel at rates proportional to their mass. TOF utilizes this variable to infer particle size and (along with the reference database and various software) composition. (B) Orbitraps utilize ion traps to identify an analyte’s mass. These ion traps consist of trapping ionized particles between an outer electrode and inner electrode and performing a Fourier transform on the charge frequency pattern to produce the ion’s mass spectrum. (C) In a Fourier-transform ion cyclotron resonance (FT-ICR) device, ions are instead trapped in a Penning trap (which uses a magnetic field to trap ions radially and an electric field to confine particles axially). Ions will rotate at their preferred frequency in packets, which produce a free induction decay (FID) charge as they pass a pair of electrodes. This FID is a time-domain spectrum, from which a frequency-domain spectrum can be extracted via a Fourier transform. Following a mass correction, the sample’s mass spectrum can be produced from the frequency-domain spectrum.