Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry.
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 a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation.
Mirzaei
Hamid
H
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Regnier
Fred
F
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis; D008099:Liver / Q000737:chemistry / Q000187:drug effects; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D009595:Nitroparaffins / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011407:Propane / Q000031:analogs & derivatives / Q000494:pharmacology; D050050:Protein Carbonylation; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D005656:Fungal Proteins; D008670:Metals; D009595:Nitroparaffins; D011506:Proteins; D012710:Serum Albumin, Bovine; C053311:biotin hydrazide; D001710:Biotin; C016617:2-nitropropane; D011407:Propane