Identification of Organophosphate Modification on Proteins Associated with the Central Nervous System
EMSL Project ID
25720
Abstract
With an increased risk of terrorist attacks involving chemical weapons, including nerve agents, there is a need to develop new and improved countermeasures for the rapid detection of exposure. After scientific review by the NIH Counterterrorism Against Chemical Threats (CounterACT) Research Network, we have been asked to accelerate the development of our prototype sensor for use by health professionals and first-line responders. We are developing a portable antibody-based micro-analytical system for the rapid diagnosis of exposure to organophosphorus (OP) nerve agents by detecting the proteins that are readily modified by organophosphates. Specifically, nerve agents are known to phosphorylate acetylcholinesterase and butyrylcholinesterase and render these enzymes inactive toward substrate regulation. Acetylcholinesterase (AChE), for example, regulates the amount of acetylcholine (ACh) present between synapses by hydrolyzing ACh ester groups when required. However, if AChE is inhibited because of an irreversible OP-modification, acetylcholine is left unchecked and the synapses continue to fire, which can lead to a complete central nervous system catastrophy and death.
In order to develop our sensor, we are incubating purified human acetylcholinesterase and human butyrylcholinesterase with different molar ratios of the organophosphates (chlorpyrifos-oxon (CPF-oxon) and diisopropylfluorophoshonate (DFP)) to generate OP-modified protein. After clean-up and isolation, we need to determine the degree and amino acid location of the modification on each type of protein in order to optimize our incubation procedure, generate homogeneous material for antibody development, and synthesizing a isotopically labeled peptide to use as an internal standard for sensor validation experiments. This information is also very valuable, since it will be the first experiments to fully characterize the degree of modification of these proteins which will aid in the development of therapeutic countermeasures (e.g., engineered prophalatics and cocktails of antidote) and help us better understand the biological interactions and translocation of modified proteins. The best way to characterize the protein modifications is to use mass spectrometry-based approaches. LC MS/MS analysis is ideally suited to show the mass shift and amino acid location with modification.
Project Details
Project type
Limited Scope
Start Date
2007-03-27
End Date
2007-04-27
Status
Closed
Released Data Link
Team
Principal Investigator