A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling

Proteomic projects are often focused on the discovery of differentially expressed proteins between control and experimental samples. Most laboratories choose the approach of running two-dimensional (2-D) gels, analyzing them and identifying the differentially expressed proteins by in-gel digestion and mass spectrometry. To date, the available stains for visualizing proteins on 2-D gels have been less than ideal for these projects because of poor detection sensitivity (Coomassie blue stain) or poor peptide recovery from in-gel digests and mass spectrometry (silver stain), unless extra destaining and washing steps are included in the protocol. In addition, the limited dynamic range of these stains has made it difficult to rigorously and reliably determine subtle differences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium-based fluorescent dye for the detection of proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels that has properties making it well suited to high-throughput proteomics projects. The advantages of SYPRO Ruby Protein Gel Stain relative to silver stain demonstrated in this study include a broad linear dynamic range and enhanced recovery of peptides from in-gel digests for matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry.