Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Abstract

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

Characterization of the Venom Proteome for the Wandering Spider, Ctenus hibernalis (Aranea: Ctenidae)

“…Venom was collected using electrostimulation with 7V of AC current, similar to previous studies [21][22][23][24]. Anesthetized individuals were placed on clamped forceps attached to an electrode.…”

Venomics: A Mini-Review

“…An example of the complexity of venom composition is shown in the reversed-phase high-performance liquid chromatography chromatogram of crude venom from the Australian funnel-web spider ( Hadronyche infensa) ( Figure 1 ). Limited quantities of venom have also hindered attempts to characterise venom components [ 12 ]. Accessing this expansive peptide reserve requires the broad implementation of multidimensional miniaturised high-throughput strategies [ 2 ].…”

Combined Peptidomic and Proteomic Analysis of Electrically Stimulated and Manually Dissected Venom from the South American Bullet Ant Paraponera clavata

“…There was also a large number of proteins in the unknown category, a recurring issue in most venomics studies. This is due to the lack of genomic and transcriptomic bioinformatics data available for a number of venoms, especially those of ants, as well as the added problem of low abundance 'masking' due to high abundance proteins 25,65,79 .…”