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

Abstract

Oxidation of proteins leads to carbonylationῌthe formation of aldehydes or ketonesῌat 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 12 C6-DNPH and 13 C6-DNPH. This method has the following steps: the oxidized protein or peptide is divided into two parts, and these parts are independently labeled with 12 C6-DNPH and 13 C6-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 di#erence 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 13 C6-DNPH provides rapid and accurate detection of carbonylated peptides even in complex mixtures.

Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective

“…Relative quantification studies using stable isotope coding allows comparing the degree of oxidation of a particular site between two or more samples. Isotopomers of DNPH [78], Girard-P reagent [32], O-ECAT [35], HICAT [79], iTRAQ [80,81], PIC reagent [82] and most recently targeted 18 O-Labeling [77] have been used in relative quantification studies of carbonylated proteins. Even though relative quantitation provides invaluable insights into the mechanisms of protein carbonylation very little is known about the fraction of any particular protein or protein site being oxidized in selected condition.…”

Protein carbonylation: Proteomics, specificity and relevance to aging

“…Two isotopic forms of Girard reagent (which contains a hydrazide group) have been used to quantify and characterize carbonylation sites in proteins, using yeast extracts treated with H 2 O 2 (Mirzaei & Regnier, ). Other reagents, such as light and heavy 13 C 6 ‐labeled‐DNPH (Kinumi et al, ), an iTRAQ hydrazide derivative named iTRAQH) (Palmese et al, ) or carbonyl‐specific element‐coded affinity mass tags (Lee et al, ) have proved useful for detecting and quantifying carbonyl content in protein samples containing a discrete number of purified proteins. However, their use in complex biological samples has not been demonstrated.…”