Protein kinases are highly evolved signal transduction enzymes that regulate almost every biological process known. As members of the largest gene family in the human genome, understanding the cellular role of each protein kinase will greatly improve our ability to selectively perturb cell signaling to treat diseases such as cancer. In the last 3-5 years, the description of interaction maps of entire organisms such as Saccharomyces cerevisiae, have provided a system wide "snapshot" of all protein protein interactions in a cell. Protein kinases have been identified in such nodes and as key components of all aspects of the interaction map. Yet, interaction maps do not address the fundamental action of each protein kinase (i.e. which substrates are phosphorylated by which kinase in the cell.) Chemical genetic tools for analysis of the direct substrates of any protein kinase in the genome have successfully identified novel downstream substrates of v-Src, CDC28, JNK, CDK2 and others. The substrate tracing approach relies on an engineered kinase, which can uniquely accept an [y-32P] ATP analog ([y-32P] A*TP). Direct substrates of the kinase of interest are identified by standard protein chromatography and analysis for 32P containing proteins. The drawback of this approach has been the difficulty in isolation of low abundance substrates or substrates phosphorylated at low stoichiometry. The vast majority of kinase substrates fall into this category, since they represent regulators of critical molecular machines, which drive the cell cycle, chromosome segregation, etc., rather than typical "house-keeping" proteins, which are often abundant. To overcome this limitation, a new method for identification of low abundance phosphoproteins is proposed. The new method relies on development of a new ATP analog capable of delivering an affinity tag to the direct substrates of any kinase. The affinity tag will function like biotin, in that it can be used as a handle to purify proteins away from irrelevant non-kinase substrate related proteins. The complete repertoire of substrates phosphorylated by the mammalian mitotic kinase CDC2/cyclin B will be analyzed by this method in order to address the fundamental role of this key kinase in driving the mitotic component of the mammalian cell cycle. These targets will provide new avenues for targeted cancer therapy. [unreadable] [unreadable]