Enrichment of Carbonylated Peptides Using Girard P Reagent and Strong Cation Exchange Chromatography

Abstract

It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of protein oxidation by a wide variety of routes is the generation of carbonyl groups. Of major interest in the study of oxidative stress diseases is which proteins in a proteome are being oxidized and the site(s) of oxidation. Based on the fact that proteins are generally characterized through tryptic peptide fragments, this paper reports a method for the isolation of oxidized peptides, which involves (1) derivatization of oxidized proteins with Girard P reagent (GRP; 1-(2-hydrazino-2-oxoethyl)pyridinium chloride), (2) following proteolysis enrichment of the derivatized peptide using strong cation exchange (SCX) chromatography, and (3) identification of oxidation sites using tandem mass spectrometry. Derivatization of aldehydes and ketones in oxidized proteins was accomplished by reacting protein carbonyls with the hydrazide of GRP. The resulting hydrazone bond was reduced by sodium cyanoborohydride to further stabilize the labeling. Derivatization time and concentrations of the derivatizing agent were optimized with model peptides. Oxidized transferrin was used as model protein to study derivatization efficiency at the protein level. Following metal-catalyzed oxidation of transferrin, the protein was derivatized with GRP and trypsin digested. Positively charged peptides were then selected from the digest with SCX chromatography at pH 6.0. Seven GRP-derivatized peptides were found to be selected from transferrin by MALDI-TOF-TOF analysis. Fourteen underivatized native peptides were also captured by the SCX column at pH 6.0. Mapping of the derivatized peptides onto the primary structure of transferrin indicated that the oxidation sites were all on solvent-accessible regions at the protein surface. Efficiency of the method was further demonstrated in the identification of oxidized proteins from yeast.