Sensitive detection of 4-hydroxy-2-nonenal-modified proteins in oxidative stress-related diseases

Protein modification by 4-hydroxy-2-nonenal (HNE) is associated with metabolic, neurodegenerative, and infectious diseases characterized by oxidative stress. Detecting low-abundance HNE-protein adducts in complex extracts requires antibodies with suitable reactivity and selectivity. We generated polyclonal antisera in rabbits using matched-species rabbit serum albumin modified in vitro with HNE (RSA-HNE), administered either intact or after tryptic digestion. The antisera displayed distinct recognition profiles across dot blot, ELISA, and Western blot assays using HNE-modified standards and membrane-protein extracts from uninfected and Plasmodium falciparum-infected human erythrocytes. Antiserum 1, raised against intact RSA-HNE, showed the stronger overall immunochemical response, whereas Antiserum 2, raised against tryptic RSA-HNE, displayed a different and more restricted peptide-recognition profile. In an immunoenrichment and LC-MS/MS workflow, Antiserum 1 recovered 14 candidate HNE-associated membrane proteins from erythrocytes infected with P. falciparum trophozoites. Site-specific evidence remained tentative for four peptides. These antisera are therefore proposed as research tools for studying protein lipoxidation, although biological replication and orthogonal validation of the candidate modification sites are required.