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.
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.
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