Abstract
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.
Highlights
The cholinesterases have been recognized as the principal targets for irreversible inhibition by organophosphate toxicants.[1]
The first goal was to determine whether monoclonal antibody depY recognizes chlorpyrifos oxon (CPO)-labeled proteins in CPOtreated HEK 293 cell lysates
This is interpreted to mean that 95 μM CPO is consumed by something other than tyrosine
Summary
The cholinesterases have been recognized as the principal targets for irreversible inhibition by organophosphate toxicants.[1]. In 1963, Sanger reported amino acid sequences in the vicinity of the labeled serine for both enzymes.[4]. These sequences were confirmed in the mid-1980s for AChE5 and for BChE.[6,7]. In their review from 1949, Koelle and Gilman stated “The fact that a compound is capable of inhibiting cholinesterase does not imply that it produces its pharmacological effects by this mechanism” alone.[1]. This concept was resurrected in the late 1990s8−11 and serves as a cornerstone for a line of investigation that we have been pursuing for the past
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
