In recent years, mass spectrometry (MS)-based phosphoproteomics have been improved, especially phosphopeptide enrichment approaches, such as immunoprecipitation with anti-phosphotyrosine antibodies 9, immobilized metal ion affinity chromatography (IMAC) 10, 11 and metal oxide chromatography with enhancers 12, 13, 14, 15. However, although genomics- and proteomics-based approaches have been applied to characterize kinases 6, 7, 8, we still lack basic information on many kinases, such as their substrates, phosphorylation motifs and molecular functions, and more information is required to unveil the entire network of protein phosphorylation. Large-scale cancer genome sequencing projects have been conducted to detect mutationally activated proteins, including kinases, in cancer signaling pathways 5, and inhibitors of oncogenic kinases have been extensively developed for cancer therapy since the success of imatinib for Bcr-Abl-positive chronic myeloid leukemia (CML) patients. It is well known that kinase-mediated phosphorylation signals are involved in various diseases such as cancer, and often cause the disease itself or drive its progression. This indicates that each protein kinase involves roughly hundreds of phosphorylation events, and the phosphorylation network formed by them must be extremely complicated.
Based on the results from human genome project, at least 518 protein kinases are considered to be encoded in human genome 2, and at least 70% of all human proteins are phosphorylated by these kinases 3, 4.
Reversible protein phosphorylation catalyzed by both protein kinases and phosphatases plays essential roles in cellular signal transduction that controls a variety of cellular functions 1.
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