Analysis of oxidative modification of proteins.

Reactions between protein molecules and reactive oxygen species (ROS) often lead to the modification of certain amino acid residues such as histidine, lysine, arginine, proline, and threonine, forming carbonyl derivatives. Carbonylation of proteins has thus often been employed for the quantification of generalized protein oxidation. Besides carbonylation, other types of oxidative damage that have been investigated in depth are the modifications of cysteine, tyrosine, and aspartate, or asparagine residues. Except for cysteine residues, whose oxidation is often determined by the loss of protein thiol groups, quantification of oxidative damage to tyrosine, and aspartate residues is usually carried out by the measurement of specific oxidation products such as dityrosine, nitrotyrosine (when nitrogen species are the oxidants), and isoaspartate. Methods described in this unit include spectrophotometry, immunoblotting, radiolabeling, GC/MS, ELISA adapted for analysis of oxidative modification.