2D DIGE Saturation Labeling for Minute Sample Amounts
The 2D DIGE technique, based on fluorophores covalently linked to amino acid side chain residues and the concept of an internal standard, has significantly improved reproducibility, sensitivity, and the dynamic range of protein quantification. In saturation DIGE, sulfhydryl groups of cysteines are labeled with cyanine dyes to completion, providing a so far unraveled sensitivity for protein detection and quantification in 2D gel-based proteomic experiments. Only a few micrograms of protein per 2D gel facilitate the analysis of about 2,000 analytes from complex mammalian cell or tissue samples. As a consequence, 2D saturation DIGE is the method of choice when only minute sample amounts are available for quantitative proteome analysis at the level of proteins rather than peptides. Since very low amounts of samples have to be handled in a reproducible manner, saturation DIGE-based proteomic experiments are technically demanding. Moreover, successful saturation DIGE approaches require a strict adherence to adequate reaction conditions at each step. This chapter is dedicated to colleagues already experienced in 2D PAGE protein separation and intends to support the establishment of this ultrasensitive technique in proteomic workgroups. We provide basic guidelines for the experimental design and discuss crucial aspects concerning labeling chemistry, sample preparation, and pitfalls caused by labeling artifacts. A detailed step-by-step protocol comprises all aspects from initial sample preparation to image analysis and statistical evaluation. Furthermore, we describe the generation of preparative saturation DIGE gels necessary for mass spectrometry–based spot identification.
Key wordsQuantification Protein Proteomics 2D DIGE Saturation labeling CyDyes
The authors would like to extend their appreciation to the Deutsche Forschungsgemeinschaft (DFG) for ongoing research grants within the research unit FOR 1041, previous grants within FOR 478 and GRK 1029, the Federal Ministry for Education and Research (BMBF) for ongoing research grants within PHANOMICS, FUGATO and FUGATO-plus (REMEDY), the European Union for a research grant within the Plurisys consortium, and the European Science Foundation (ESF) for funding of the “Stressflea” consortium. The authors wish to acknowledge their appreciation to Patrick Bolbrinker, Myriam Demant, Daniela Deutsch, and Julia Korte for critical reading of the manuscript and to Miwako Kösters for technical assistance.
- 3.Sitek, B., Sipos, B., Alkatout, I., Poschmann, G., Stephan, C., Schulenborg, T., Marcus, K., Luttges, J., Dittert, D. D., Baretton, G., Schmiegel, W., Hahn, S. A., Kloppel, G., Meyer, H. E., and Stuhler, K. (2009) Analysis of the Pancreatic Tumor Progression by a Quantitative Proteomic Approach and Immunhistochemical Validation, J Proteome Res., 4, 1647–56.CrossRefGoogle Scholar
- 5.Sitek, B., Potthoff, S., Schulenborg, T., Stegbauer, J., Vinke, T., Rump, L. C., Meyer, H. E., Vonend, O., and Stuhler, K. (2006) Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling, Proteomics 6, 4337–45.PubMedCrossRefGoogle Scholar
- 6.Helling, S., Schmitt, E., Joppich, C., Schulenborg, T., Mullner, S., Felske-Muller, S., Wiebringhaus, T., Becker, G., Linsenmann, G., Sitek, B., Lutter, P., Meyer, H. E., and Marcus, K. (2006) 2-D differential membrane proteome analysis of scarce protein samples, Proteomics 6, 4506–13.PubMedCrossRefGoogle Scholar
- 7.Berendt, F. J., Frohlich, T., Bolbrinker, P., Boelhauve, M., Gungor, T., Habermann, F. A., Wolf, E., and Arnold, G. J. (2009) Highly sensitive saturation labelling reveals changes in abundance of cell cycle-associated proteins and redox enzyme variants during oocyte maturation in vitro, Proteomics 9, 550–64.PubMedCrossRefGoogle Scholar
- 9.Fröhlich T. and Arnold G.J. (2011) Quantifying Attomole Amounts of Proteins from Complex Samples by Nano-LC and Selected Reaction Monitoring (SRM), Methods Mol Biol., 790 141–164.Google Scholar