Mass spectrometry method to identify aging pathways of Sp- and Rp-tabun adducts on human butyrylcholinesterase based on the acid labile P-N bond.
The phosphoramidate nerve agent tabun inhibits butyrylcholinesterase (BChE) and acetylcholinesterase by making a covalent bond on the active site serine. The adduct loses an alkyl group in a process called aging. The mechanism of aging of the tabun adduct is controversial. Some studies claim that aging proceeds through deamination, whereas crystal structure studies show aging by O-dealkylation. Our goal was to develop a method that clearly distinguishes between deamination and O-dealkylation. We began by studying the tetraisopropyl pyrophosphoramide adduct of BChE because this adduct has two P-N bonds. Mass spectra showed that the P-N bonds were stable during trypsin digestion at pH 8 but were cleaved during pepsin digestion at pH 2. The P-N bond in tabun was also acid labile, whereas the P-O bond was stable. A scheme to distinguish aging by deamination from aging by O-dealkylation was based on the acid labile P-N bond. BChE was inhibited with Sp- and Rp-tabun thiocholine nerve agent model compounds to make adducts identical to those of tabun with known stereochemistry. After aging and digestion with pepsin at pH 2, peptide FGES198AGAAS from Sp-tabun thiocholine had a mass of 902.2 m/z in negative mode, indicating that it had aged by deamination, whereas peptide FGES198AGAAS from Rp-tabun thiocholine had a mass of 874.2 m/z in negative mode, indicating that it had aged by O-dealkylation. BChE inhibited by authentic, racemic tabun yielded both 902.2 and 874.2 m/z peptides, indicating that both stereoisomers reacted with BChE and aged either by deamination or dealkylation.
Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950, USA.
Wei
W
Jiang
John R
JR
Cashman
Florian
F
Nachon
Patrick
P
Masson
Lawrence M
LM
Schopfer
Oksana
O
Lockridge
D002091:Butyrylcholinesterase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009584:Nitrogen / Q000378:metabolism*; D010758:Phosphorus / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010758:Phosphorus; D002091:Butyrylcholinesterase; D009584:Nitrogen