Abstract |
The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE), which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography--tandem mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODE-modified proteins were located by 2D sodium dodecyl sulfate-- polyacrylamide gel electrophoresis and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress.
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Authors | Peter G Slade, Michelle V Williams, Viral Brahmbhatt, Ajit Dash, John S Wishnok, Steven R Tannenbaum |
Journal | Chemical research in toxicology
(Chem Res Toxicol)
Vol. 23
Issue 3
Pg. 557-67
(Mar 15 2010)
ISSN: 1520-5010 [Electronic] United States |
PMID | 20131800
(Publication Type: Journal Article, Research Support, N.I.H., Extramural)
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Chemical References |
- 9,12-dioxo-10-dodecenoic acid
- Fatty Acids, Monounsaturated
- Linoleic Acids
- Lipid Peroxides
- Proteins
- 13-hydroperoxy-9,11-octadecadienoic acid
- Cytochromes c
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Topics |
- Cell Line, Tumor
- Chromatography, Liquid
- Cytochromes c
(metabolism)
- Fatty Acids, Monounsaturated
(metabolism)
- Humans
- Linoleic Acids
(metabolism)
- Lipid Peroxidation
- Lipid Peroxides
(metabolism)
- Proteins
(analysis, metabolism)
- Tandem Mass Spectrometry
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