On-substrate Enzymatic Reaction to Determine Acetylcholinesterase Activity in Whole Blood by Paper Spray Mass Spectrometry

Daniel O Carmany,Jennifer W Sekowski,Paul S Demond,Ethan M Mcbride,Phillip M Mach,Bernard Benton,Michael W Busch,Gabrielle M Rizzo,Elizabeth S Dhummakupt,Trevor Glaros

doi:10.1007/s13361-018-2072-1

Daniel O Carmany, Jennifer W Sekowski + Show 8 more

Open Access

https://doi.org/10.1007/s13361-018-2072-1

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Abstract

Currently, all assays measuring acetylcholinesterase (AChE) activity following a suspected nerve agent exposure leverage methodologies that fail to identify the agent. This limits the overall effectiveness and ability to administer proper countermeasures. As such, there is an urgent need to identify novel, rapid, and more comprehensive approaches to establish AChE activity, including identification of the toxicant. Paper spray mass spectrometry was used to monitor the activity of acetylcholinesterase, both in-solution and on modified hydrophobic paper surface. Hydrophobic paper surfaces were prepared using vaporized trichloro(3,3,3-trifluoropropyl)silane. In both approaches, mixtures of diluted human whole blood with and without VX were mixed with a non-endogenous AChE specific substrate, 1,1-dimethyl-4-acetylthiomethylpiperidinium (MATP+). Formation of the cleaved MATP+ product was monitored over time and compared to MATP+ to determine relative AChE activity. This on-substrate assay was effective at determining AChE activity and identifying the toxicant; however, determination of AChE activity in-solution proceeded at a slower rate. The on-substrate assay serves as a pioneering example of an enzymatic reaction occurring on the surface of a paper spray ionization ticket. This work broadens the range of applications relating to paper spray ionization-based clinical diagnostic assays.Graphical ᅟ

Highlights

Acetylcholine (ACh) is the most widespread and best understood neurotransmitter
Carmany et al.: Enzyme Reaction with Blood by Paper Spray Mass Spectrometry organophosphate (OP) pesticides, this leads to nerve signal transmission dysfunction
MATP+ was selected for use as the AChE substrate due to a high specificity for the enzyme when compared to the BChE enzyme

Summary

Introduction

The primary clinical reason for measuring AChE or BChE activity is to diagnosis human exposure to pesticides, such as organophosphates (OPs) and carbamates, which act as cholinesterase inhibitors. These assays are based upon the products which are created when AChE/BChE cleaves its target substrate. In order to establish the level of inhibition of both enzymes, two separate assays must be performed, typically with the inclusion of a BChE-specific inhibitor while measuring AChE activity.

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Conclusion