Selection and identification of oxidized proteins and their oxidation sites

Hamid Mirzaei, Purdue University


The goal of this research was to develop methods for selection and identification of carbonylated proteins and their oxidation sites. The work reported here began with the research for development of a method for the isolation of oxidized proteins, which involved (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. Using this method 14 peptides and their parent proteins were identified from tryptic digest of oxidized proteins from rat liver homogenates. Next a faster, more targeted method was developed to allow selection and identification of carbonylated petides from the whole proteome which involved (1) labeling oxidized proteins with 1-(Carboxymethyl)pyridinium chloride (GPR) and (2) enrichment of the labeled peptide using strong cation exchange chromatography followed by identification of oxidation sites using tandem mass spectrometry. This research was followed by development of a more advanced approach which allowed better separation and higher resolution of oxidized proteins. The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase chromatography (RPC) on a C8 column. Slightly over 400 proteins were identified.




Regnier, Purdue University.

Subject Area

Analytical chemistry

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server