title: Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry.
abstract: Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we developed an immunoaffinity purification and liquid chromatography-mass spectrometry (LC-MS) strategy for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. Using a purified BChE protein, we initially identified the exact phosphorylation site on the serine residue (S198) with a 108 Da modification by both MS/MS and accurately measured parent ion masses and quantified the extent of phosphorylation on S198 following paraoxon treatment to be >99.9%. Then, the phosphorylated BChE peptide in paraoxon-treated human plasma following immunoaffinity purification was successfully identified based on the accurate measured mass and retention time information initially obtained from the purified BChE protein. Thus, immunoaffinity purification combined with LC-MS represents a viable approach for the detection and quantification of phosphorylated BChE as an exposure biomarker of organophosphates and nerve agents.
authors.affiliation: Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Uma K
authors.initials: UK
authors.lastname: Aryal
authors.forename: Chiann-Tso
authors.initials: CT
authors.lastname: Lin
authors.forename: Jong-Seo
authors.initials: JS
authors.lastname: Kim
authors.forename: Tyler H
authors.initials: TH
authors.lastname: Heibeck
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
authors.forename: Yuehe
authors.initials: Y
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D055910:Antibodies, Immobilized / Q000276:immunology; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002091:Butyrylcholinesterase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D053801:Capillary Electrochromatography; D002846:Chromatography, Affinity; D006801:Humans; D007163:Immunosorbent Techniques; D008280:Magnetics; D008969:Molecular Sequence Data; D010261:Paraoxon / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D055910:Antibodies, Immobilized; D010748:Phosphopeptides; D001710:Biotin; D019809:Streptavidin; D002091:Butyrylcholinesterase; D010261:Paraoxon