Mass spectrometric characterizations of protein carbonylation: Comparison between three different conditions, oxidation by CuII/ascorbic acid, adduction of methyl glyoxal, and adduction of 4-hydroxy-2(E)-nonenal

• Protein carbonylation reactions were examined by mass spectrometry.
• Reactions of three different types of carbonylation were compared.
• Carbonylation occured efficiently in the presence of ROS and lipids.

Oxidative stress can cause various protein modifications, mainly carbonylation. There are three major chemical ways to introduce hydrazine reactive carbonyls: (i) direct oxidation by reactive oxygen species (ROS), (ii) adduction of sugars or sugar-related aldehydes, and (iii) adduction of lipid-related aldehydes. Here, we performed a comparative study of three representative conditions: CuII/ascorbic acid (AA) (ROS), methylglyoxal (sugar-related), and 4-hydroxy-2(E)-nonenal (HNE) (lipid-related). Insulin β chain and Girard’s reagent P were used as the model protein and hydrazine reagent, respectively. LC/MS analyses of reactions with HNE or CuII/AA-treated linoleic acid revealed that protein carbonylation under oxidative stress is the most efficient in the presence of polyunsaturated fatty acids.

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