Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment
| Autor: | Thomas A. Blake, Rudolph C. Johnson, Lawrence M. Schopfer, Oksana Lockridge, Seda Onder, Ozden Tacal |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
medicine.drug_class Blotting Western Enzyme-Linked Immunosorbent Assay Toxicology Monoclonal antibody Serine Mice 03 medical and health sciences Capillary electrophoresis Western blot Tandem Mass Spectrometry Liquid chromatography–mass spectrometry medicine Animals Humans Amino Acid Sequence Tyrosine Peptide sequence Molecular Structure biology medicine.diagnostic_test Chemistry Antibodies Monoclonal Proteins General Medicine HEK293 Cells 030104 developmental biology Biochemistry Proteolysis biology.protein Chlorpyrifos Antibody Peptides Chromatography Liquid |
| Zdroj: | Chemical Research in Toxicology. 31:520-530 |
| ISSN: | 1520-5010 0893-228X |
| DOI: | 10.1021/acs.chemrestox.8b00083 |
| Popis: | Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 beta, and alpha-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure. |
| Databáze: | OpenAIRE |
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