Background

Protein phosphorylation is one of the most intensively studied post translation modifications. Protein phosphorylation acts as an “on” or “off” switch for the target protein activity and thereby modulates a large number of pathways and biological processes (Hunter, 1995). Phosphorylation of proteins is reversibly mediated by the action of two classes of enzymes namely; protein kinases and protein phosphatases, which together constitute the largest enzyme family, spanning 2% of the human genome (Venter, 2001; Manning et al., 2002; Alonso et al., 2004). It is estimated that one in three proteins undergoes phosphorylation during its lifetime. Given the scale of impact and importance of protein phosphorylation in dictating protein function, tight regulation of phosphorylation by protein kinases and phosphatases play vital role in signal transduction, cell differentiation, development, cell cycle control and metabolism (Delom and Chevet, 2006; Ardito et al., 2017). Protein kinases can be divided into two major groups based on the site of phosphorylation; serine/threonine kinases and tyrosine kinases (Roskoski, 2015). Whereas up to 86.4% of all phosphorylation modifications occur at serine residues, followed by 11.8% at threonine residues, less than 2% occur at tyrosine residues (Ardito et al., 2017).

The analysis of phospho-proteins is coupled with a myriad number of complications. Majority of proteins exist as a heterogeneous population, wherein the phosphorylated fraction is generally low in abundance and it exists in several different phosphorylated forms. The reversible nature of phosphorylation due to activity of phosphatases further necessitates crucial precautions during the processing of samples (Delom and Chevet, 2006). Mass spectroscopy analysis enables identification and quantification of post-translation modifications of proteins at a large scale (Steen et al., 2006; Junger and Aebersold, 2014; Pan et al., 2015). Two-dimensional gel electrophoresis can exploit the difference in isoelectric point of phosphorylated variant of a protein to quantify phosphorylated fraction (Guy et al., 1994). Use of phospho-protein specific antibodies conjugated with fluorophores allows single cell based rapid quantification with the help of flow cytometry (Krutzik and Nolan, 2003). Flow cytometry offers reduction in the number of processing steps as compared to western blotting and enables multiplex analysis of different types of cells and phospho-proteins (Krutzik et al., 2004; Davies et al., 2016). Whereas traditional biochemical assays can only quantify phosphorylation in the bulk population of cells but cannot determine the signaling differences in a sub-population within a heterogeneous population of cells, flow cytometry can couple cell surface marker expression data with phosphorylation data to understand differential response of different cell types to a ligand or inhibitor (Krutzik et al., 2004). Phospho-protein flow cytometry can be used to study signaling pathways in stem cells (Sonowal et al., 2013), cancer cells (Kumar et al., 2017) and understand cell-cell interaction (Kumar et al., 2018). We developed a reliable method to analyze phospho-proteins in adherent cells. Adherent cells require an additional step, where the cells have to be enzymatically detached from the cell growth surface and can lead to inactivation of phospho-proteins. This protocol described here can be used to successfully quantify the intracellular phospho-proteins in adherent cell types.