Many highly reactive, toxic chemicals will react with proteins in blood to form chemical-protein adducts. Recently there has been interest to understand and utilize chemical-protein adducts for discovering novel mechanisms of toxicity and for biomonitoring reactive environmental chemicals. Identifying and quantifying chemical-protein adducts remains challenging. We are developing a multipronged approach to identify protein adducts caused by environmental chemicals. As a chemical model, we used dose-dependent concentrations of chlorpyrifos oxon (bioactive metabolite of the pesticide chlorpyrifos) to form adducts in purified proteins, plasma, and whole blood. First, global mass spectrometry proteomic techniques were used to identify chemical-protein adducts. Samples were digested into peptides with trypsin and separated into 12 fractions using high-pH reverse-phase chromatography. Each fraction was analyzed using a 100 min low-pH reverse-phase LC coupled to an Orbitrap Velos mass spectrometer for high resolution MS/MS analysis. A custom bioinformatics workflow was utilized to generate confident chemical-protein adduct identifications. A new MS visualization tool was developed to aid in validation of chemical-protein adducts from MS/MS data. Second, activity-based protein profiling was used to enrich potential sites of adduct formation. After synthesizing adducts, samples were treated with a fluorophosphonate (FP2) probe to enrich target sites of chlorpyrifos oxon binding. SDS-PAGE was used to visually confirm competitive binding of the probe and chlorpyrifos oxon, and mass spectrometry was used to identify potential sites for adduct formation. This multipronged strategy identified multiple protein adducts on serine and tyrosine amino acids on several proteins including complement C3, hemoglobin B1, histone H3, and epsilon globin 1. We anticipate that this new approach can be broadly applied to identify chemical-protein adducts for discovering novel mechanisms of toxicity and biomonitoring toxic chemicals that are traditionally difficult to monitor.<br/><br/>Supported by PNNL Laboratory Directed Research.

Identifying chemical-protein adducts using a multipronged approach

254th National Meeting (2017)

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