Phosphoprotein Enrichment for Protein Analysis in Motile T-Lymphocytes

Protein phosphorylation plays a key role in intracellular signal transduction and regulates diverse cellular functions. This posttranslational modification of proteins occurs dynamically and reversibly and only a small fraction of the total proteins is phosphorylated at any given time depending on the cell types and their functioning. Thus, a relatively low abundance of phosphorylated proteins is present in specific cells under certain conditions and hence it becomes problematic to detect these proteins and their analysis. In particular, phosphoproteomic analysis of rapidly migrating T-lymphocytes is always challenging. In order to analyze phosphoproteins in motile T-cells using techniques such as polyacrylamide gel electrophoresis and mass spectrometry, it is often important to enrich the phosphorylated forms in the cellular lysates. In this chapter, we describe a simple method to enrich phosphoproteins that can be used for protein analysis in motile T-cells.