Proteomics identification of oxidatively modified proteins in brain.
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.
Sultana
Rukhsana
R
Department of Chemistry, Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055, USA.
Perluigi
Marzia
M
Butterfield
D Allan
DA
D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D010659:Phenylhydrazines / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010659:Phenylhydrazines; C004787:2,4-dinitrophenylhydrazine