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.
Keywords: Crosslink; Crosslinks; Dichlorvos; Isopeptide; Manual evaluation; Mass spectrometry; Protein Prospector; SH-SY5Y human Neuroblastoma cells.
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