title: Analysis and sequencing of the active-site peptide from native and organophosphate-inactivated acetylcholinesterase by electrospray ionization, quadrupole/time-of-flight (QTOF) mass spectrometry.
abstract: A method to identify and sequence recombinant mouse acetylcholinesterase (rMoAChE) including the native and organophosphate-modified active-site peptides was developed using capillary liquid chromatography with electrospray ionization, quadrupole/time-of-flight mass spectrometry. Addition of 2-propanol to the reversed-phase gradient system and a decreased gradient slope improved the peptide resolution and the signal of the active-site peptide. The highest protein coverage and active-site peptide signal were achieved when the rMoAChE:chymotrypsin ratio of 5:1 was used with digestion at 37 degrees C. rMoAChE and the active-site peptide were identified and sequenced from chymotryptic digests of native, methyl paraoxon-, and ethyl paraoxon-inactivated rMoAChE showing unequivocally that the exact modification site was the active-site serine.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, Center for Structural and Functional Neuroscience, The University of Montana, Missoula, 59812, USA.
authors.forename: Reggie S
authors.initials: RS
authors.lastname: Spaulding
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: George
authors.forename: Charles M
authors.initials: CM
authors.lastname: Thompson
mesh_terms: D000110:Acetylcholinesterase / Q000187:drug effects*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002853:Chromatography, Liquid / Q000379:methods*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002800:Cholinesterase Inhibitors; D010455:Peptides; D000110:Acetylcholinesterase