Proteome profiling of aging in mouse models: differential expression of proteins involved in metabolism, transport, and stress response in kidney.

Aging is a time-dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age-associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19-22 wk) and old (24 months) C57/BL6 male mice using 2-DE followed by LC-MS/MS. We found that expression levels of 49 proteins were upregulated (p < or = 0.05), while that of only ten proteins were downregulated (p < or = 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase beta-subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig-binding protein, low density lipoprotein receptor-related protein associated protein 1, selenium-binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase beta subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age-related kidney degeneration.

Comparison of SYPRO Ruby and Deep Purple using commonly available UV transilluminator: wide-scale application in proteomic research.

Fluorescent stains are becoming increasingly useful in proteomics research involving protein expression as well as post-translational modification studies and are particularly useful for samples which are expensive and scarce. The fluorescent dyes Deep Purple and SYPRO Ruby are widely used in protein expression studies. Using UV transillumination and Charged Coupled Device (CCD) based imaging system, their relative sensitivity to detect proteins separated by two-dimensional polyacrylamide gel electrophoresis and downstream protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was compared. Using mouse liver homogenate, we detected a greater number of spots using SYPRO Ruby over Deep Purple stain. However, the number of matched peptides and the percentage of amino acid residues identified for 21 different proteins were comparable suggesting their equivalency for LC-MS/MS identification. In spite of comparable MS compatibility, we recommend the use of SYPRO Ruby for expression proteomics due to its higher sensitivity in detecting protein spots.

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins.

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.