@article{aea67c724498487c99ae3d5b00fd8312,
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",
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.",
keywords = "N2a neuroblastoma, Protein Prospector, SH-SY5Y, diethoxyphosphotyrosine, mass spectrometry, rabbit monoclonal, single B cell cloning",
author = "Seda Onder and {Van Grol}, Marco and Alex Fidder and Gaoping Xiao and Daan Noort and Udaya Yerramalla and Ozden Tacal and Schopfer, {Lawrence M.} and Oksana Lockridge",
note = "Funding Information: The authors thank Dr. Robert Chalkley, University of California San Francisco, for helpful advice on the use of Protein Prospector. Protein Prospector programs are available at no cost, https://prospector.ucsf.edu . Programs were developed in the University of California San Francisco Mass Spectrometry Facility, directed by Dr. Alma Burlingame, funded by the NIH National Institute for General Medical Sciences. The Proteomics Toolkit http://db.systemsbiology.net:8080/proteomicsToolkit/ , Proteome Discoverer (Thermo Scientific, Waltham, MA), and XCalibur Qual Browser (Thermo Scientific, Waltham, MA) were used to identify ions in MS/MS spectra. Mass spectrometry data were obtained by the Mass Spectrometry and Proteomics Core Facility at the University of Nebraska Medical Center, which is supported by state funds from the Nebraska Research Initiative. Funding Information: Supported by the Fred & Pamela Buffet Cancer Center Support Grant P30CA036727, NIH grant 1R21ES030132-01A1 (to O.L.), and TUBITAK grant SBAG-318S259 (to S.O.). Funding Information: The authors thank Dr. Robert Chalkley, University of California San Francisco, for helpful advice on the use of Protein Prospector. Protein Prospector programs are available at no cost, https://prospector.ucsf.edu. Programs were developed in the University of California San Francisco Mass Spectrometry Facility, directed by Dr. Alma Burlingame, funded by the NIH National Institute for General Medical Sciences. The Proteomics Toolkit http://db.systemsbiology.net:8080/proteomicsToolkit/, Proteome Discoverer (Thermo Scientific, Waltham, MA), and XCalibur Qual Browser (Thermo Scientific, Waltham, MA) were used to identify ions in MS/MS spectra. Mass spectrometry data were obtained by the Mass Spectrometry and Proteomics Core Facility at the University of Nebraska Medical Center, which is supported by state funds from the Nebraska Research Initiative. Supported by the Fred & Pamela Buffet Cancer Center Support Grant P30CA036727, NIH grant 1R21ES030132-01A1 (to O.L.), and TUBITAK grant SBAG-318S259 (to S.O.). Publisher Copyright: {\textcopyright} 2021 The Authors. Published by American Chemical Society.",
year = "2021",
doi = "10.1021/acs.jproteome.1c00383",
language = "English (US)",
journal = "Journal of proteome research",
issn = "1535-3893",
publisher = "American Chemical Society",
}