TY - JOUR
T1 - Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment
AU - Onder, Seda
AU - Schopfer, Lawrence M.
AU - Tacal, Ozden
AU - Blake, Thomas A.
AU - Johnson, Rudolph C.
AU - Lockridge, Oksana
N1 - Funding Information:
*E-mail: [email protected]. ORCID 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, Defense Threat Reduction Agency 11-005-12430 (to T.A.B. and R.C.J.), and TUBITAK (BIDEB-2211A) fellowship (to S.O.). 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.
Funding Information:
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 Defense Threat Reduction Agency 11-005-12430 (to T.A.B. and R.C.J.) and TUBITAK (BIDEB-2211A) fellowship (to S.O.).
Publisher Copyright:
Copyright © 2018 American Chemical Society 2018.
PY - 2018/6/18
Y1 - 2018/6/18
N2 - Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 β, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.
AB - Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 β, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.
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U2 - 10.1021/acs.chemrestox.8b00083
DO - 10.1021/acs.chemrestox.8b00083
M3 - Article
C2 - 29775289
AN - SCOPUS:85047413570
SN - 0893-228X
VL - 31
SP - 520
EP - 530
JO - Chemical Research in Toxicology
JF - Chemical Research in Toxicology
IS - 6
ER -