High-Throughput Proteomic-Based Identification of Oxidatively Induced Protein Carbonylation in Mouse Brain

Purpose. The major initiative of this study was to implement a novel proteomic approach in order to detect protein carbonylation in aged mouse brain. Several lines of evidence indicate that reactive oxygen species (ROS)-induced protein oxidation plays an essential role in the initiation of age-related neuropathologies. Therefore, the identification of free radical or peroxide substrates would provide further insight into key biochemical mechanisms that contribute to the progression of certain neurological disorders. Methods. Historically, ROS targets have been identified by conventional immunological two-dimensional (2-D) gel electrophoresis and mass spectrometric analyses. However, specific classes of proteins, such as transmembrane-spanning proteins, high-molecular-weight proteins, and very acidic or basic proteins, are frequently excluded or underrepresented by these analyses. In order to fill this technologic gap, we have used a functional proteomics approach using a liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis coupled with a hydrazide biotin-streptavidin methodology in order to identify protein carbonylation in aged mice. Results. Our initial studies suggest an ability to identify at least 100 carbonylated proteins in a single LC-MS/MS experiment. In addition to high-abundance cytosolic proteins that have been previously identified by 2-D gel electrophoresis and mass spectrometric analyses, we are able to identify several low-abundance receptor proteins, mitochondrial proteins involved in glucose and energy metabolism, as well as a series of receptors and tyrosine phosphatases known to be associated with insulin and insulin-like growth factor metabolism and cell-signaling pathways. Conclusions. Here we describe a rapid and sensitive proteomic analysis for the identification of carbonylated proteins in mouse brain homogenates through the conjunction of liquid chromatography and tandem mass spectrometry methods. We believe the ability to detect these post-translationally modified proteins specifically associated with brain impairments during the course of aging should allow one to more closely and objectively monitor the efficacy of various clinical treatments. In addition, the discovery of these unique brain biomarkers could also provide a conceptual framework for the future design of alternative drugs in the treatment of a variety of age-related neurodegenerative disorders.