Summary
The two-dimensional (2-D) gel-based proteomics platform remains the workhorse for proteomics and is fueled by a number of key improvements, including fluorescence-based stains for detection and quantification of proteins and phosphoproteins with high sensitivity and linear dynamic ranges. One such stain is Pro-Q diamond phosphoprotein stain (Pro-Q DPS), which binds to the phosphate moiety of phospho-proteins irrespective of the phosphoamino acid. We recently introduced a modified Pro-Q DPS protocol to detect phosphoprotein spots on 2-D gels with very low background addressing some prime concerns, including high cost and reproducibility of Pro-Q DPS. The major modifications were a threefold dilution of Pro-Q DPS and the use of threefold less volume of the diluted staining solution. In this chapter, use of the modified Pro-Q DPS protocol along with the 2-D gel-based proteomics for phosphoprotein detection and quantification is described in detail. This 2-D gel- and Pro-Q DPS-based proteomics workflow has seven major steps: preparation of total protein, separation of proteins by 2-D gel electrophoresis, detection of phosphoprotein and total protein, image analysis and quantitative expression profiling, excision of 2-D spots, mass spectrometry analysis, and data processing and organization. Involvement of the modified Pro-Q DPS protocol in this proteomics workflow alone reduces the overall cost by at least ninefold for conducting phospho-proteomics analysis on a global scale, thereby making this entire process economically attractive to the scientific community.
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Acknowledgments
This research was supported by National Science Foundation Plant Genome Research grants DBI-0332418 and DBI-0445287 (to J.J.T.). The authors have no conflicts-of-interest with the manufacturer of Pro-Q DPS, other mentioned products, or equipment.
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Agrawal, G.K., Thelen, J.J. (2009). A High-Resolution Two Dimensional Gel- and Pro-Q DPS-Based Proteomics Workflow for Phosphoprotein Identification and Quantitative Profiling. In: Graauw, M.d. (eds) Phospho-Proteomics. Methods in Molecular Biology™, vol 527. Humana Press. https://doi.org/10.1007/978-1-60327-834-8_1
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DOI: https://doi.org/10.1007/978-1-60327-834-8_1
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