Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry

Abstract

Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant ( K d 3.2 × 10 –8 M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 μM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.

Chlorpyrifos Oxon Activates Glutamate and Lysine for Protein Cross-linking

“…As it turns out, in 1962, Sanger showed that the equivalent position in human albumin, tyrosine-411, was labeled by diisopropylphosphate . Additional work has shown that proteins form covalent adducts on tyrosine with a variety of OP compounds. − We also found that OP makes covalent bonds with the side chain of lysine. ,,, …”

“…We are certain that CPO adds diethyl phosphate to the side chain of tyrosine in proteins. This certainty comes from use of a highly selective antidiethyl phosphate-tyrosine antibody to detect and immunopurify OP-tyrosine-modified proteins and peptides . MS/MS spectra support OP-tyrosine adducts, refer to and Figure S2.…”

“…This certainty comes from use of a highly selective antidiethyl phosphate-tyrosine antibody to detect and immunopurify OP-tyrosine-modified proteins and peptides . MS/MS spectra support OP-tyrosine adducts, refer to and Figure S2. Tables and show a relatively high abundance of diethylphospho-tyrosine adducts.…”

Selective cell retrieval method using light-responsive gas-generating polymer-based microarrays

“…For example, monoclonal antibodies have been developed by a signal analysis of antigen-specific single B-cells using a microarray chip to collect B-cells, analyze, and clone antibody genes. 4,5 In recent years, research for detecting circulating tumor cells (CTC) has been conducted by various methods. 6 For example, microarray and microfluidic devices have been used to determine the ratio of CTC in a blood sample.…”

An Aptasensor Based on a Flexible Screen-Printed Silver Electrode for the Rapid Detection of Chlorpyrifos

“…Several biosensors characterized by different transducing mechanisms, for example, electrochemical, optical, or mass-based, have been applied for the detection of CPF during the last decades [ 14 , 17 , 18 ]. Among all, the electrochemical method has recently become an attractive choice because of its quick and highly sensitive response, coupled with the ability to integrate with a wide range of biorecognition elements such as aptamers, enzymes, or antibodies easily [ 19 ].…”