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Reversible phosphorylation of proteins controlled by kinases and phosphatases is one of the most ubiquitous post-translational modifications, and regulates a variety of cell functions through cellular signal transduction pathways. These signals are involved in various diseases such as cancer and rheumatism, and often cause the disease itself or drive the progression. Quantitative phosphoproteomics based on liquid chromatography-tandem mass spectrometry combined with phosphopeptide enrichment and stable isotope labeling allows profiling thousands of phosphorylation sites on human proteins and has been applied to monitoring cellular phosphorylation dynamics induced by various growth factors, hormones and other perturbations including kinase inhibitors. Here, we employed these technologies to quantify the temporal response of phosphorylation dynamics in SKBR3 breast cancer cells to lapatinib, a kinase inhibitor for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 μM lapatinib. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.

Protein phosphorylation, one of the most ubiquitous post-translational modifications of proteins, is reversibly controlled by protein kinases and phosphatases, and regulates a variety of cell functions through cellular signal transduction pathways. These signals are involved in various diseases such as cancer, and often cause the disease itself or its progression. Phosphoproteomics has been explored to analyze protein phosphorylation comprehensively and has been applied to monitoring the entire network of cellular signal transduction. In this review, the frontlines of phosphoproteomics as well as its application to cancer molecular targeting drug discovery, therapy and diagnostics are described.

We developed two methods for solubility screening of drug candidates in drug discovery. The first is a solution-precipitation (SP) method, in which the sample solutions are prepared by adding the drug solution in dimethylsulfoxide (DMSO) to buffers followed by filtering off the precipitate using 96-well filterplate. The second is a powder-dissolution (PD) method, in which the solid samples are dissolved to the buffer in the HPLC vial equipped with the filter membrane in the HPLC autosampler. An HPLC equipped with a photodiode array detector is used to measure the concentration of the sample solutions in both methods. The SP method was used for high throughput screening the solvating process of the candidates in aqueous solutions with lower sample consumption, and the PD method was used for screening both inter-molecular interaction in solid state and solvation in aqueous solution with more sample amount than that of SP method. Therefore, the solubility screening from early to final stage of lead optimization process would be successfully accomplished by using both methods complementarily.