Identification of proteins modified by the lipid peroxidation aldehyde DODE in MCF7 breast cancer cells.

摘要

The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic (DODE) which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE and verified the usefulness of DODE as a biomarker of oxidative stress. To reduce false positives, three methods were use 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 2D-LC-MS/MS. Second, cells were treated with native 13-HPODE. Protein-carbonyls were biotinylated with an aldehyde reactive probe (ARP) and modified proteins enriched by neutravidin affinity and identified by 2D-LCMS/MS. Third, using a newly developed DODE antibody, DODE modified proteins were located by 2D-SDS-PAGE and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods demonstrated a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells.