Comparative gel-based phosphoproteomics in response to signaling molecules.

The gel-based proteomics approach is a valuable technique for studying the characteristics of proteins. This technique has diverse applications ranging from analysis of a single protein to the study of the total cellular proteins. Further, protein quality and to some extent distribution can be first assessed by means of one-dimensional gel electrophoresis and then more informatively, for comparative analysis, using the two-dimensional gel electrophoresis technique. Here, we describe how to take advantage of the availability of fluorescent dyes to stain for a selective class of proteins on the same gel for the detection of both phospho- and total proteomes. This enables the co-detection of phosphoproteins as well as total proteins from the same gel and is accomplished by utilizing two different fluorescent stains, the ProQ-Diamond, which stains only phosphorylated proteins, and Sypro Ruby, which stains the entire subset of proteins. This workflow can be applied to gain insights into the regulatory mechanisms induced by signaling molecules such as cyclic nucleotides through the quantification and subsequent identification of responsive phospho- and total proteins.