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Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells

Chlorpyrifos oxon catalyzes the crosslinking of proteins via an isopeptide bond between lysine and glutamic acid or aspartic acid in studies with purified proteins. Our goal was to determine the crosslinking activity of the organophosphorus pesticide, dichlorvos. We developed a protocol for examinin...

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Autores principales: Schopfer, Lawrence M., Lockridge, Oksana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10368009/
https://www.ncbi.nlm.nih.gov/pubmed/35961399
http://dx.doi.org/10.1016/j.ab.2022.114844
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author Schopfer, Lawrence M.
Lockridge, Oksana
author_facet Schopfer, Lawrence M.
Lockridge, Oksana
author_sort Schopfer, Lawrence M.
collection PubMed
description Chlorpyrifos oxon catalyzes the crosslinking of proteins via an isopeptide bond between lysine and glutamic acid or aspartic acid in studies with purified proteins. Our goal was to determine the crosslinking activity of the organophosphorus pesticide, dichlorvos. We developed a protocol for examining crosslinks in a complex protein mixture consisting of human SH-SY5Y cells exposed to 10 μM dichlorvos. The steps in our protocol included immunopurification of crosslinked peptides by binding to anti-isopeptide antibody 81D1C2, stringent washing of the immobilized complex, release of bound peptides from Protein G agarose with 50% acetonitrile 1% formic acid, liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer, Protein Prospector searches of mass spectrometry data, and manual evaluation of candidate crosslinked dipeptides. We report a low quantity of dichlorvos-induced KD and KE crosslinked proteins in human SH-SY5Y cells exposed to dichlorvos. Cells not treated with dichlorvos had no detectable KD and KE crosslinked proteins. Proteins in the crosslink were low abundance proteins. In conclusion, we provide a protocol for testing complex protein mixtures for the presence of crosslinked proteins. Our protocol could be useful for testing the association between neurodegenerative disease and exposure to organophosphorus pesticides.
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spelling pubmed-103680092023-07-25 Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells Schopfer, Lawrence M. Lockridge, Oksana Anal Biochem Article Chlorpyrifos oxon catalyzes the crosslinking of proteins via an isopeptide bond between lysine and glutamic acid or aspartic acid in studies with purified proteins. Our goal was to determine the crosslinking activity of the organophosphorus pesticide, dichlorvos. We developed a protocol for examining crosslinks in a complex protein mixture consisting of human SH-SY5Y cells exposed to 10 μM dichlorvos. The steps in our protocol included immunopurification of crosslinked peptides by binding to anti-isopeptide antibody 81D1C2, stringent washing of the immobilized complex, release of bound peptides from Protein G agarose with 50% acetonitrile 1% formic acid, liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer, Protein Prospector searches of mass spectrometry data, and manual evaluation of candidate crosslinked dipeptides. We report a low quantity of dichlorvos-induced KD and KE crosslinked proteins in human SH-SY5Y cells exposed to dichlorvos. Cells not treated with dichlorvos had no detectable KD and KE crosslinked proteins. Proteins in the crosslink were low abundance proteins. In conclusion, we provide a protocol for testing complex protein mixtures for the presence of crosslinked proteins. Our protocol could be useful for testing the association between neurodegenerative disease and exposure to organophosphorus pesticides. 2022-10-15 2022-08-09 /pmc/articles/PMC10368009/ /pubmed/35961399 http://dx.doi.org/10.1016/j.ab.2022.114844 Text en https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Article
Schopfer, Lawrence M.
Lockridge, Oksana
Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells
title Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells
title_full Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells
title_fullStr Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells
title_full_unstemmed Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells
title_short Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells
title_sort dichlorvos-induced formation of isopeptide crosslinks between proteins in sh-sy5y cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10368009/
https://www.ncbi.nlm.nih.gov/pubmed/35961399
http://dx.doi.org/10.1016/j.ab.2022.114844
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