Mass spectrometric compatibility of Deep Purple and SYPRO Ruby total protein stains for high-throughput proteomics using large-format two-dimensional gel electrophoresis Christina M. Nock 1 y , Malcolm S. Ball 2 y , Ian R. White 1 , J. Mark Skehel 1 , Louisa Bill 1 and Peter Karuso 2,3 * 1 GlaxoSmithKline Pharmaceuticals, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK 2 FLUOROtechnics Pty Ltd., Macquarie University, Sydney NSW 2109, Australia 3 Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia Received 25 November 2007; Revised 31 January 2008; Accepted 3 February 2008 In order to identify putative biomarkers from two-dimensional (2D) gel electrophoresis it is necessary to use a visualization technique that is sensitive, has a large dynamic range and does not interfere with the identification of the protein. As mass spectrometry increases in sensitivity more pressure is placed on visualization techniques that facilitate proteomic workflows but do not interfere with downstream processing. Two stains reported to meet these requirements are SYPRO Ruby (Invitrogen) and Deep Purple (GE Healthcare). This study examined the compatibility of these stains with protein identification by selecting spots from replicate 2D gels of human plasma and subjecting these to protein identification using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using a test of two populations of proportions it was found that proteins were statistically more likely to be identified from gels stained with Deep Purple. Additionally, the identifications from Deep Purple stained gels are of higher quality because they are based on multiple peptides. Copyright # 2008 John Wiley & Sons, Ltd. Two-dimensional polyacrylamide gel electrophoresis (2DE) is one of the most widely used techniques in proteomics today. The methodology is well understood, provides highly visual, analog data with equipment available for automated running, staining and analysis of multiple gels. Accordingly, it is well suited to relatively high-throughput analysis of samples and identification of putative biomarkers in research and clinical settings. For example, 2DE has been applied successfully for identification of tumor markers 1 and identification of individuals susceptible to diabetes. 2 The challenge in biomarker discovery is that complex biological samples, such as plasma, may contain proteins in a wide range of concentrations exceeding 10 orders of magnitude 3 and the desired biomarker may be present in low abundance. For this reason protein stains must be sensitive and have sufficient dynamic range to allow determination of concen- tration changes of potential biomarkers. In practice this can best be achieved with radioisotope or fluorescence tech- niques, because colorimetric stains, such as Coomassie or silver, are either not sensitive enough for modern proteomics or have too small a dynamic range. 4–6 The safety concerns with use of radioisotopes have resulted in replacement of this technique with fluorescence in recent years. While a 2DE gel generates a large amount of high- resolution analog data in a single experiment, proteins with expression differences contributing to spot intensity changes must be identified using techniques such as peptide mass fingerprinting (PMF), peptide mapping (high-performance liquid chromatography/tandem mass spectrometry (HPLC/ MS/MS)), Edman sequencing, 7 or immunochemical staining. Mass spectrometric techniques are particularly useful for protein identification from 2DE gels as they lend themselves to automation and high-throughput methodologies. 8,9 The sensitivity of mass spectrometers has enabled proteins to be detected at femtomole to attomole levels from pure samples which means that, in theory, any protein visualized on a 2D gel should be detectable by mass spectrometry. 10 However, in practice, interference from the gel matrix, sample handling and stain interference conspire with protein sequence specific issues to reduce realistic detection limits to the mid-to-high femtomolar level. 11 The ability of 2D gels to be used as a diagnostic tool therefore relies on three characteristics: (1) that the detection method is sensitive enough to detect proteins present in the sample with sufficient dynamic range to allow quantification of proteins over a broad range of concentrations; (2) that the variability of the detection method is less than any real change in expression levels of the protein of interest; and RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 881–886 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3483 *Correspondence to: P. Karuso, Department of Chemistry & Bio- molecular Sciences, Macquarie University, Sydney NSW 2109, Australia. E-mail: peter.karuso@mq.edu.au y These authors contributed equally to this work. Copyright # 2008 John Wiley & Sons, Ltd.