A high-resolution two dimensional Gel- and Pro-Q DPS-based proteomics workflow for phosphoprotein identification and quantitative profiling.

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.