Identification of nitrated proteins from iNOS-induced macrophages using biotin labeling and capture

TOXI 75

Jennifer R. Seal, jseal@mit.edu, Biological Engineering Division, MIT, 77 Massachusetts Ave, Cambridge, MA 02139, John S. Wishnok, Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, and Steven R. Tannenbaum, Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 56-731A, Cambridge, MA 02139.
Tyrosine nitration is an important post-translational modification that is associated with oxidative stress and activation of nitric oxide synthases, leading to increased production of nitric oxide. Described here is a method that combines specific isolation and enrichment of nitrated proteins from complex biological systems. Proteins modified with nitrotyrosine were reduced to their corresponding aminotyrosine followed by biotin coupling. Biotinylated proteins were then enriched by avidin capture followed by 2D gel electrophoresis. Proteins were identified by mass spectrometric methods including confirmation of structure by peptide mass fingerprinting and LC/MS/MS, with data analysis by MASCOT and Spectrum Mill. This method was applied to the analysis of macrophages activated to produce nitric oxide which, through formation of other reactive nitrogen species, leads to tyrosine nitration.
 

Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster

Sci-Mix
8:00 PM-10:00 PM, Monday, August 20, 2007 BCEC -- Exhibit Hall - B2, Sci-Mix

Division of Chemical Toxicology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007