title: High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase.
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 <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >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.
authors.affiliation: Battelle at the Centers for Disease Control and Prevention , Atlanta, Georgia 30341, United States.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Mathews
authors.affiliation: Centers for Disease Control and Prevention , National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia 30341, United States.
authors.forename: Melissa D
authors.identifier: 0000-0002-8530-0795
authors.initials: MD
authors.lastname: Carter
authors.affiliation: Oak Ridge Institute for Science and Education Fellow at the Centers for Disease Control and Prevention , Atlanta, Georgia 30341, United States.
authors.forename: Darryl
authors.initials: D
authors.lastname: Johnson
authors.affiliation: Centers for Disease Control and Prevention , National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia 30341, United States.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Isenberg
authors.affiliation: Battelle at the Centers for Disease Control and Prevention , Atlanta, Georgia 30341, United States.
authors.forename: Leigh Ann
authors.initials: LA
authors.lastname: Graham
authors.affiliation: Centers for Disease Control and Prevention , National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia 30341, United States.
authors.forename: Jerry D
authors.initials: JD
authors.lastname: Thomas
authors.affiliation: Centers for Disease Control and Prevention , National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia 30341, United States.
authors.forename: Rudolph C
authors.initials: RC
authors.lastname: Johnson
mesh_terms: D015415:Biomarkers / Q000097:blood; D002091:Butyrylcholinesterase / Q000097:blood / Q000737:chemistry / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D005188:False Negative Reactions; D005189:False Positive Reactions; D006801:Humans; D047468:Immunoprecipitation; D000067397:Nerve Agents / Q000592:standards / Q000633:toxicity*; D009842:Oligopeptides / Q000097:blood* / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000592:standards / Q000633:toxicity*; D011786:Quality Control; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards
chemical_list: D015415:Biomarkers; D000067397:Nerve Agents; D009842:Oligopeptides; D009943:Organophosphorus Compounds; D002091:Butyrylcholinesterase