Proteins Modified by the Lipid Peroxidation Aldehyde 9,12-Dioxo-10(E)-dodecenoic Acid in MCF7 Breast Cancer Cells

• Main Authors: Slade, Peter G. (Contributor), Williams, Michelle V. (Contributor), Brahmbhatt, Viral (Contributor), Wishnok, John S. (Contributor), Dash, Ajit (Contributor), Tannenbaum, Steven Robert (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Chemistry (Contributor), Tannenbaum, Steven R. (Contributor)
• Format: Article
• Language: English
• Published: American Chemical Society, 2011-11-02T20:21:05Z.
• Subjects: Article
• Online Access: Get fulltext

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.
Harvard University--MIT Division of Health Sciences and Technology
United States. National Institutes of Health (NCI Program Project Grant CA26731)
National Institute of Environmental Health Sciences (Grant P30 ES002109)