Oxime blot: A novel method for reliable and sensitive detection of carbonylated proteins in diverse biological systems

Oxidative stress and oxidative protein damage occur in various biological processes and diseases. The carbonyl group on amino acid side chains is the most widely used protein oxidation biomarker. Carbonyl groups are commonly detected indirectly through their reaction with 2,4-dinitrophenylhydrazine (DNPH) and subsequent labeling with an anti-DNP antibody. However, the DNPH immunoblotting method lacks protocol standardiza-tion, exhibits technical bias, and has low reliability. To overcome these shortcomings, we have developed a new blotting method in which the carbonyl group reacts with the biotin-aminooxy probe to form a chemically stable oxime bond. The reaction speed and the extent of the carbonyl group derivatization are increased by adding a p-phenylenediamine (pPDA) catalyst under neutral pH conditions. These improvements are crucial since they ensure that the carbonyl derivatization reaction reaches a plateau within hours and increases the sensitivity and robustness of protein carbonyl detection. Furthermore, derivatization under pH-neutral conditions facilitates a good SDS-PAGE protein migration pattern, avoids protein loss by acidic precipitation, and is directly compatible with protein immunoprecipitation. This work describes the new Oxime blot method and demonstrates its use in detecting protein carbonylation in complex matrices from diverse biological samples.

Automated summary

Oxidative stress and oxidative protein damage commonly occur in biological processes and diseases. The carbonyl group on amino acid side chains is a widely used biomarker for protein oxidation. However, the current DNPH immunoblotting method has limitations in standardization, technical bias, and reliability. To address these issues, a new blotting method using a biotin-aminooxy probe and p-phenylenediamine catalyst for carbonyl group derivatization under neutral pH conditions has been developed. This method improves the sensitivity and robustness of protein carbonyl detection and is compatible with protein immunoprecipitation.