2D gel proteomics: an approach to study age-related differences in protein abundance or isoform complexity in biological samples.

This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat.

S-adenosylmethionine prevents chronic alcohol-induced mitochondrial dysfunction in the rat liver.

An early event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes to the mitochondrial proteome, respiration defects, and mitochondrial DNA (mtDNA) damage. S-adenosylmethionine (SAM) has emerged as a potential therapeutic for treating alcoholic liver disease through mechanisms that appear to involve decreases in oxidative stress and proinflammatory cytokine production as well as the alleviation of steatosis. Because mitochondria are a source of reactive oxygen/nitrogen species and a target for oxidative damage, we tested the hypothesis that SAM treatment during alcohol exposure preserves organelle function. Mitochondria were isolated from livers of rats fed control and ethanol diets with and without SAM for 5 wk. Alcohol feeding caused a significant decrease in state 3 respiration and the respiratory control ratio, whereas SAM administration prevented these alcohol-mediated defects and preserved hepatic SAM levels. SAM treatment prevented alcohol-associated increases in mitochondrial superoxide production, mtDNA damage, and inducible nitric oxide synthase induction, without a significant lessening of steatosis. Accompanying these indexes of oxidant damage, SAM prevented alcohol-mediated losses in cytochrome c oxidase subunits as shown using blue native PAGE proteomics and immunoblot analysis, which resulted in partial preservation of complex IV activity. SAM treatment attenuated the upregulation of the mitochondrial stress chaperone prohibitin. Although SAM supplementation did not alleviate steatosis by itself, SAM prevented several key alcohol-mediated defects to the mitochondria genome and proteome that contribute to the bioenergetic defect in the liver after alcohol consumption. These findings reveal new molecular targets through which SAM may work to alleviate one critical component of alcohol-induced liver injury: mitochondria dysfunction.