Identifying the substrates of protein kinases remains a major obstacle in the elucidation of eukaryotic signaling pathways. Promiscuity among kinases and their substrates coupled with the extraordinary plasticity of phosphorylation networks renders traditional genetic approaches or small-molecule inhibitors problematic when trying to determine the direct substrates of an individual kinase. Here we describe methods to label, enrich, and identify the direct substrates of analogue-sensitive kinases by exploiting their steric complementarity to artificial ATP analogues. Using calcium-dependent protein kinases of Toxoplasma gondii as a model for these approaches, this protocol brings together numerous advances that enable labeling of kinase targets in semi-permeabilized cells, quantification of direct labeling over background, and highly specific enrichment of targeted phosphopeptides.
AS kinase; IMAC; LC MS/MS; Quantitative analysis; SILAC; Toxoplasmosis