Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1.
Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.
Gade
Dörte
D
Max Planck Institute for Marine Microbiology, Bremen, Germany.
Thiermann
Jürgen
J
Markowsky
Dieter
D
Rabus
Ralf
R
D000117:Acetylglucosamine; D001419:Bacteria / Q000737:chemistry* / Q000254:growth & development; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005063:Europium; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D005947:Glucose; D012680:Sensitivity and Specificity; D012984:Software; D013194:Staining and Labeling
D001426:Bacterial Proteins; D003470:Culture Media; C436863:SYPRO Rose Plus; D005063:Europium; D005947:Glucose; D000117:Acetylglucosamine