AcomparisonofsilverstainandSYPRORuby ProteinGelStainwithrespecttoproteindetection intwo-dimensionalgelsandidentificationby peptidemassprofiling Proteomic projects are often focused on the discovery of differentially expressed pro- teins between control and experimental samples. Most laboratories choose the approach of running two-dimensional (2-D) gels, analyzing them and identifying the dif- ferentially 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 destain- ing 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 dif- ferences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium- based fluorescent dye for the detection of proteins in sodium dodecyl sulfate-polyacryl- amide 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. Keywords: Protein stains / Two-dimensional gel electrophoresis / Mass spectrometry / Fluores- cence / Mass profiling / Proteomics / Ruthenium EL 4189 MaryF.Lopez 1 KieraBerggren 2 ElenaChernokalskaya 3 AlexanderLazarev 1 MyraRobinson 1 WayneF.Patton 2 1 Proteome Systems, Inc., Woburn, MA, USA 2 Molecular Probes, Inc., Eugene, OR, USA 3 Vertex Pharmaceuticals Cambridge, MA, USA 1 Introduction As the human genome sequencing effort reaches its con- clusion, the focus in the pharmaceutical industry is begin- ning to shift towards the proteins that actually carry out functional activities. The quantitative measurement of gene expression at the protein level, referred to as pro- teome analysis, complements molecular biology ap- proaches such as DNA sequencing, subtractive hybridiza- tion, differential display, and microarray assays of gene expression in addressing complex biological problems. Two-dimensional gel electrophoresis followed by mass spectrometry of excised protein spots has rapidly become the operating paradigm for proteome analysis [1±11]. Many laboratories are focused on processing multiple samples in a high-throughput manner to enable the rapid identification of differentially expressed protein targets for drug or diagnostic discovery. Two limiting factors for this type of complete proteomic mapping are the sensitivity of the staining technology and the recovery of peptides from in-gel digests for mass spectrometric identification. There are several approaches to enhancing spot detection sen- sitivity, among them sample prefractionation and using more sensitive stains [12]. Until recently, the most sensi- tive stains available have been alkaline/silver diamine and acidic/silver nitrate stains [13, 14]. Unfortunately, these stains are also the most destructive for mass spectrome- try because many of the fixation techniques include glu- taraldehyde, which can cross-link the proteins and pre- vent efficient digestion by trypsin or other proteases [15, 16]. Though glutaraldehyde can be omitted from silver stain formulations to improve compatibility with mass spectrometry, detection sensitivity is compromised and peptide yields are often lower than those obtained from Coomassie blue-stained proteins unless extra laborious destaining and washing steps are included in the protocol [15±17]. SYPRO  Ruby Protein Gel Stain was recently developed as a sensitive fluorescent stain for proteomic applications and has been shown to have several advan- tages over other commonly used protein stains [17]. In this report, we compare SYPRO Ruby Protein Gel stain to an acidic/silver nitrate stain specifically with respect to sensitivity, dynamic range and recovery of peptides from in-gel digests for matrix-assisted laser desorption/ioniza- tion mass spectrometry. Correspondence: Mary F. Lopez, PhD, Proteome Systems Inc., 25 Industrial Dr., Chelmsford, MA 01824, USA E-mail: psiconsult@earthlink.net Fax: +781-275-9652 Abbreviation:MBB, monobromobimane Electrophoresis 2000, 21, 3673±3683 3673  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/1717-3673 $17.50+.50/0 Proteomicsand2-DE