Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase.
Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30 min to purify BChE from 1 ml plasma. The method uses a microfuge spin column to build a 0.2 ml procainamide affinity column. The eluted BChE contains 3-4 microg of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.
Li
He
H
Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA. heli@berkeley.edu
Tong
Larry
L
Schopfer
Lawrence M
LM
Masson
Patrick
P
Lockridge
Oksana
O
D000595:Amino Acid Sequence; D002091:Butyrylcholinesterase / Q000097:blood* / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000378:metabolism*; D011342:Procainamide / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010755:Organophosphates; D002091:Butyrylcholinesterase; D011342:Procainamide