Abstract
Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.
Highlights
In the 1970s and 1980s William Catterall and colleagues discovered that isolated scorpion venom components bound with high affinity to ion channels
The venom was a complex mixture of molecules with a high percentage of proteinaceous materials ranging from 3–6 kDa with an average venom yield from 50 collections of ~148 ± 12 μg of total protein per μL of venom as determined by conventional Bradford assay
100 μg of lyophilized venom from each species of Poecilotheria were suspended in 1 mL of the initial mobile phase and a 1 μL aliquot injection was made into an Ultra Performance Liquid Chromatograph (UPLC) coupled to a HDMS
Summary
In the 1970s and 1980s William Catterall and colleagues discovered that isolated scorpion venom components bound with high affinity to ion channels. Their investigations led to discovery of the voltage-gated sodium channel (VGSC) complex [1,2]. From these early studies, we know that there are nine isoforms of VGSCs (denoted Nav 1.1–1.9) that initiate and propagate the rising phase of an action potential [3]. Gain of function mutations in VGSCs have been shown to participate
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