Purpose:
Nerve agents, including as sarin, tabun, and soman, are classified as chemical warfare agents and represent a class of organophosphate compounds similar to many commonly used pesticides. Upon exposure, nerve agents prevent deactivation of acetylcholine signaling to muscles resulting in miosis, glandular hypersecretion, and death in doses as small as 10 mg. The ability to rapidly detect and quantify exposure to these agents is critical for medical response and patient care.
Currently used methods for detecting nerve agent exposure exist for both urine and serum samples. These methods are either slow or rely on the use of active agent precluding their use in standard laboratories.
We have developed a rapid method for the detection and quantitation of V and G series nerve agent exposure in serum that does not require the use of live agent. Identification of specific nerve agent adducts and unadducted proteins is possible using this method and there are no interferences from organophosphate pesticides.
Methods:
Adducted and nonadducted butyrylcholinesterase was extracted from 0.5 mL human serum using affinity chromatography. Peptides were produced by on-bead digestion with pepsin and the reaction was stopped by 10 kDa molecular weight filtration. The resulting peptide sample was separated on a capillary HPLC and analyzed on a triple quad mass spectrometer using multiple reaction monitoring.
Results:
Butyrylcholinesterase can be successfully isolated from serum and digested into peptide fragments in less than 5 hours using this method. Approximately 1 mg of protein is extracted from the serum sample constituting roughly 20% of the expected total butyrylcholinesterase in the sample. Pepsin digestion yields peptide fragments that can be analyzed on a tandem mass spectrometer with an LOD of l.2 ng/mL. Both unadducted and adducted peptides can be individually chromatographed and quantitated using this method so that percent inhibitions can be calculated for exposure to GB, GF, VX, and RVX.
Conclusions:
We have developed a rapid, robust, and reliable method for detecting and quantitating nerve agent exposure in serum samples by isolating butyrylcholinesterase and monitoring peptides for the presence of specific adducts. This method provides identification of the nerve agent without interferences from organophosphate pesticides. Quantitation of both adducted and nonadducted peptides provides a mechanism for calculating the percent inhibition of sera thereby addressing variations in butyrylcholinesterase levels between individuals. |
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