Several studies demonstrated the involvement of
free radicals in the pathophysiology of
neurodegenerative diseases. Once formed,
reactive oxygen species (ROS) can promote multiple forms of oxidative damage, including
protein oxidation, and thereby influence the function of a diverse array of cellular processes leading inevitably to neuronal dysfunctions.
Protein oxidation can therefore rapidly contribute to oxidative stress by directly affecting cell signaling, cell structure, and enzymatic processes such as metabolism. There are many different modes of inducing
protein oxidation including
metal-catalyzed oxidation, oxidation-induced cleavage of
peptide chain,
amino acid oxidation, and the covalent binding of lipid peroxidation products or advanced glycation end proteomics. In this paper we describe the protocol of redox proteomics, a tool to identify post-translational modifications of
proteins. We focus our attention on the identification of carbonylated and 4-hydroxy-2-trans-nonenal-bound
proteins. In redox proteomics, samples for the identification of
protein carbonyls are first derivatized with 2,4-dinitrophenolhydrazine (DNPH) followed by two-dimensional (2D) separation of these
proteins based on their isoelectric point and rate of migration. The carbonylated
proteins are then detected using 2D Western blot techniques. Similarly, HNE-bound
proteins can be detected using the above-mentioned strategy except that the sample does not need to be derivatized. Separated
proteins are identified following tryptic digestion, mass spectrometry, and interrogation of appropriate databases.