Protein Carbonylation Detected with Light and Heavy Isotope-Labeled 2,4-Dinitrophenylhydrazine by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

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Oxidation of proteins leads to carbonylation—the formation of aldehydes or ketones-at the amino acid side chain and/or the terminal amino groups. Carbonylated proteins have been conventionally detected by UV absorption spectrometry of the stable adduct with 2,4-dinitrophenylhydrazine (DNPH). However, this routine method is limited to detection of the total carbonyl content and does not provide structural information. We developed an isotope-dilution method for the specific detection of carbonylated proteins using ¹²C₆-DNPH and ¹³C₆-DNPH. This method has the following steps: the oxidized protein or peptide is divided into two parts, and these parts are independently labeled with ¹²C₆-DNPH and ¹³C₆-DNPH; the mixtures of these two labeled solutions are subsequently measured with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The carbonylated peptide was found by searching for a doublet peak having a mass difference of 6 Da. We examined oxidized angiotensin I and oxidized lysozyme prepared by treatment with NaOCl. The oxidized angiotensin I showed four pairs of doublet peaks in the MALDI-TOF mass spectrum. The structure was determined by tandem mass spectrometry. In the case of tryptic digest of the oxidized lysozyme, two carbonylated products could be easily identified even in a complex mixture. The use of ¹³C₆-DNPH provides rapid and accurate detection of carbonylated peptides even in complex mixtures.