Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research.

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.

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.