Article pubs.acs.org/ac
High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase Thomas P. Mathews,† Melissa D. Carter,*,‡ Darryl Johnson,§ Samantha L. Isenberg,‡ Leigh Ann Graham,† Jerry D. Thomas,‡ and Rudolph C. Johnson‡ †
Battelle at the Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia 30341, United States § Oak Ridge Institute for Science and Education Fellow at the Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States ‡
S Supporting Information *
ABSTRACT: In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with 15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.
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rganophosphorus nerve agents (OPNAs)1,2 are a class of internationally banned chemical warfare agents that broadly target a family of enzymes known as serine hydrolases.3 OPNAs disable serine hydrolases by phosphorylating their active site serine residues. Since OPNAs have such broad inhibitory activity, many different combinations of ligands retain potent activity. Therefore, over a thousand4 distinct species of OPNA structures have been defined as schedule 1 agents by the Chemical Weapons Convention.5 Inhibition of acetylcholinesterase (AChE) in the nervous system by OPNAs is responsible for producing a number of characteristic toxic effects including seizures and associated motor convulsions. AChE inhibition leads to accumulation of acetylcholine in synapses and a loss of signal transmission.2 Traditionally, inhibition of AChE was detected using the © 2017 American Chemical Society
Ellman activity assay which measures choline production as a measure of enzyme activity.6 This assay has been widely used for clinical assays, such as the detection of organophosphorus (OP) pesticide exposure.7 While effective, activity-based assays cannot identify a specific agent in an exposure event. Hydrolyzed OPNA metabolites identify metabolites of specific agents from urine or blood;8,9 however, these metabolites are short-lived, and the majority of excretion occurs days after an exposure has occurred.10,11 Analysis of OPNA adducts to albumin offer another route to verify exposure.12 However, Received: November 10, 2016 Accepted: January 11, 2017 Published: January 24, 2017 1955
DOI: 10.1021/acs.analchem.6b04441 Anal. Chem. 2017, 89, 1955−1964
Article
Analytical Chemistry
Figure 1. Adduction mechanism of OPNAs to BChE. OPNAs inhibit their target serine hydrolases (SHs), such as BChE, by covalently binding to the active site serine. Aging of OPNA adducts occurs in blood whereby alkyl-phosphate variable groups (blue) on the OPNA adduct are nonspecifically hydrolyzed to the corresponding phosphonic or phosphoric acid while the alkyl-phosphonate variable groups (red) remain unchanged. OPCW-CWC nomenclature defines R1 as a variable alkyl group (i.e., ethyl, methyl, propyl, or cyclohexyl) or a hydroxyl group; R2 can be either a variable alkyl group or an amino alkyl group.
occur. In this case, a targeted method would not detect exposure because the precursor ion mass-to-charge ratio (m/z) would be unknown. Previous studies used product ion scans in methods with conventional triple quadrupole instruments to screen for exposure.30 However, their targeted design and low resolving power limited identification to only anticipated precursor ions. Proteomics offers an efficient route to screen for characteristic BChE nonapeptide product ions. Data-dependent highresolution tandem mass spectrometry (ddHRMS/MS) methods, most often used to sequence peptides, collect data in two distinct layers. First, the instrument acquires a full mass spectrum with high resolving power and identifies the most intense ions in that scan. Next, the instrument selects the most intense ions for dissociation, collecting high-resolution spectra on the product ions generated and tying them to specific precursor ions.31 Applying this analytical strategy to the OPNAadducted nonapeptide would provide an effective route to screen for OPNA-BChE biomarkers by their signature b-ions and identify precursor ions with high mass accuracy. Ideally, this method would complement existing targeted methodologies using the same preparation, vastly increasing laboratory efficiency. For the first time, this study describes the development, characterization, and validation of a BChE screening method capable of identifying unknown OPNA adducts to BChE through the analysis of signature product ions by UHPLCddHRMS/MS. High mass accuracy was observed across 23 independent quality control (QC) preparations of human plasma exposed to various OPNAs. Using these samples for characterization, precursor ions were identified with a mean mass accuracy of
