Extraction of α-neurotoxins from equine plasma by receptor based affinity purification.

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α-neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled "Conotoxin", was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α-neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one-step affinity purification method was developed for the detection of α-neurotoxins in plasma.

Sensitive Detection of α-Conotoxin GI in Human Plasma Using a Solid-Phase Extraction Column and LC-MS/MS.

α-conotoxin GI, a short peptide toxin in the venom of Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029-0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene-divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent (methanol: acetonitrile = 50:50, v/v). GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18 (50 × 2.1 mm, 5 µm) column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6-66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.

Stabilization of α-conotoxin AuIB: influences of disulfide connectivity and backbone cyclization.

α-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability. AuIB is a unique member of the α-conotoxin family because the nonnative "ribbon" disulfide isomer exhibits enhanced activity at the nAChR in rat parasympathetic neurons compared with the native "globular" isomer. Here we show that the ribbon isomer of AuIB is also more resistant to disulfide scrambling, despite having a nonnative connectivity and flexible structure. This resistance to disulfide scrambling does not correlate with overall stability in serum because the ribbon isomer is degraded in human serum more rapidly than the globular isomer. Cyclization via the joining of the N- and C-termini with peptide linkers of four to seven amino acids prevented degradation of the ribbon isomer in serum and stabilized the globular isomers to disulfide scrambling. The linker length used for cyclization strongly affected the relative proportions of the disulfide isomers produced by oxidative folding. Overall, the results of this study provide important insights into factors influencing the stability and oxidative folding of α-conotoxin AuIB and might be valuable in the design of more stable antagonists of nAChRs.

Rapid detection of conotoxin SO(3) in serum using Cu-chelated magnetic beads coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A novel method based on Cu-chelated magnetic beads (Cu-Magbeads) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the rapid detection of peptide toxins in serum. The peptides in the serum were efficiently adsorbed by the Cu-Magbeads, eluted with methanol solution, and assayed by MALDI-TOF-MS. Specific peptides were identified according to their characteristic mass-to-charge ratio values. Conotoxin SO(3), a synthesized peptide, was used as a model to evaluate the method. Conotoxin SO(3) was detected in human serum, as well as bovine and murine serum, with a detection sensitivity in the low femtomole range. The assay was performed within 40 min, without the need for a specific antibody or an expensive reagent. It shows potential for future use in clinical and emergency rescue practice because of its simplicity, high speed, and high sensitivity.