Analysis of Protein Carbonyls with 2,4-Dinitrophenyl Hydrazine and Its Antibodies by Immunoblot in Two-Dimensional Gel Electrophoresis

Abstract

Protein carbonyls are reported to increase in aging and in pathologies such as Alzheimer's disease and ischemic injury. Detailed study of this important issue has, however, been hampered by lack of an appropriate method to identify individual carbonylated proteins. We describe here an immunoblot method to identify individual carbonylated proteins. We describe here an immunoblot method to investigate protein carbonyls reactive to 2,4-dinitrophenyl hydrazine. Rabbit polyclonal antibodies against 2,4-dinitrophenyl hydrazine were used to study the proteins derivatized by the reagent in one- or two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting. More than 25 proteins with high carbonyl contents were clearly demonstrated in two-dimensional immunoblot of rat tissue soluble proteins. The method could detect concentrations as low as 1 pmol of carbonyls. The signals were mostly abolished by prior treatment of tissue proteins with sodium borohydride to reduce carbonyls. Fragments generated by V8 protease digestion of a single protein exhibited signal intensities of varying degrees, indicating that carbonylation is not uniform in different amino acid sequences. Proteins treated with glucose or aldehydes gave rise to positive signals, suggesting that the finding of carbonyls in tissue proteins is not necessarily an indication of direct oxidation of side chains of amino acid residues.

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis

“…Protein carbonylation was detected by western blotting for DNPH-modified proteins separated by 2D-gel electrophoresis. 24,31 This method detected significant increases in protein carbonylation in HL60 cells after 1-h VP16 treatment (Figure 3b). Figure 3c shows that pretreatment with the antioxidant NAC reduced VP16-induced protein carbonylation to control levels (i), demonstrating that, in this case, carbonylation is a result of ROS and it most likely is not the result of glycation.…”

“…However, recent advances in 2D-gel electrophoresis and proteomics have led to renewed interest in the field as it becomes possible to identify the proteins that are modified in this way. [23][24][25] A number of interesting papers have shown that during ageing, protein oxidation in response to ROS are not random, but that some proteins are more susceptible than others. 26 In the case of drosophilae flight muscle, only aconitase and adenine nucleotide translocase showed any increase in carbonylation with age, 26 and in mouse plasma ageing-associated protein oxidation was only seen in two proteins, albumin and transferrin.…”

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

“…Most of these methods are based on the Schiff base chemistry that is possible with the carbonyl group. The most traditional Schiff base method relied on the 2D Western analysis of DNPHderivatized carbonyl proteins (283) and had early applications in the identification of oxidized proteins in AD brain (74). Using shotgun proteomics methods, tryptic peptides are separated with strong cation exchange (SCX) and/or reversephase (RP) LC and detected by MS.…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…Alternatively, ELISA assays have been developed for the quantitation of DNPH-derivatised carbonyls [85][86][87]. The same chemistry can be used in combination with gel electrophoresis, followed by an immunodetection [88,89]. Alternatively, Yoo and Regnier [90] have developed a biotinylation strategy for the specific labelling of carbonylated proteins after 2-DE.…”