TY - JOUR
T1 - Use of Hupresin to Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure
AU - Onder, Seda
AU - Schopfer, Lawrence M.
AU - Cashman, John R.
AU - Tacal, Ozden
AU - Johnson, Rudolph C.
AU - Blake, Thomas A.
AU - Lockridge, Oksana
N1 - Funding Information:
*Phone: 402 559 6032. E-mail: [email protected]. ORCID Thomas A. Blake: 0000-0001-8536-9998 Oksana Lockridge: 0000-0002-8345-3640 Funding Supported by DLS/NCEH/CDC Contract 200-2015-87939 (to O.L.) and Fred and Pamela Buffett Cancer Center Support Grant P30CA036727. Centers for Disease Control and Prevention, Office of Public Health Preparedness and Response, and Defense Threat Reduction Agency 11-005-12430 (to T.A.B. and R.C.J.). Notes The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services. The authors declare no competing financial interest.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2018/1/2
Y1 - 2018/1/2
N2 - Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.
AB - Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.
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U2 - 10.1021/acs.analchem.7b04160
DO - 10.1021/acs.analchem.7b04160
M3 - Article
C2 - 29172437
AN - SCOPUS:85037586885
SN - 0003-2700
VL - 90
SP - 974
EP - 979
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 1
ER -