Title
Rabbit antidiethoxyphosphotyrosine antibody, made by single B cell cloning, detects chlorpyrifos oxon-modified proteins in cultured cells and immunopurifies modified peptides for mass spectrometry
Author
Onder, S.
van Grol, M.
Fidder, A.
Xiao, G.
Noort, D.
Yerramalla, U.
Tacal, O.
Schopfer, L.M.
Lockridge, O.
Publication year
2021
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 (Kd 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.
Subject
Diethoxyphosphotyrosine
Mass spectrometry
Protein prospector
N2a neuroblastoma
SH-SY5Y
Single B cell cloning
Rabbit monoclonal
Acetylcholinesterase, 9000-81-1
Chlorpyrifos, 2921-88-2
Acetylcholinesterase
Chlorpyrifos
O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate
Organophosphorus compounds
Peptides
Pesticides
Animal
B lymphocyte
Cell culture
Degenerative disease
Liquid chromatography
Molecular cloning
Mouse
Tandem mass spectrometry
Mice
Neurodegenerative diseases
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DOI
https://doi.org/10.1021/acs.jproteome.1c00383
TNO identifier
958873
Publisher
American Chemical Society
ISSN
1535-3893
Source
Journal of Proteome Research, 20 (20), 4728-4745
Document type
article