Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)-dodecenoic acid in MCF7 breast cancer cells.
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.
Department of Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Peter G
PG
Slade
Michelle V
MV
Williams
Viral
V
Brahmbhatt
Ajit
A
Dash
John S
JS
Wishnok
Steven R
SR
Tannenbaum
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D045304:Cytochromes c / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism*; D006801:Humans; D008041:Linoleic Acids / Q000378:metabolism; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
C418762:9,12-dioxo-10-dodecenoic acid; D005229:Fatty Acids, Monounsaturated; D008041:Linoleic Acids; D008054:Lipid Peroxides; D011506:Proteins; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D045304:Cytochromes c