Application Of On-line Sample Preconcentration Techniques Of Capillary Electrophoresis In Detection Of Protein Kinase Activity

The protein phosphorylation catalyzed by protein kinase is a kind of post-translational modification process which is transferring the y-phospate group from ATP to the hydroxyl group of a serine, theronine, or tyrosine residue of a target protein. It plays an important role in regulating intercellular signal transduction and cellular processes (such as cell growth, differentiation, proliferation, apoptosis). In eukaryotic cells,1-3% of all genes are encoded for protein kinases, while the over-expression and aberrant activity of protein kinase are closely associated with various human diseases such as inflammation, cancer, diabetes, Alzheimer's disease and so on. So, the detection of a given protein kinase was not only beneficial to promote the process of basic life science research, but also valuable to understand the disease related fundamental biological processes and plays an important role in the development of biological diagnosis therapy and anti proliferation drugs.Kinase activity has traditionally been assayed by using radiometric methods or immunoassays which are based on antibody-antigen specific action, but both two methods have shortcomings that cannot be ignored. The radiometric methods might pose great threats to human health due to hazardous radioactive reagents and immunoassays are basically cost-consuming and lack of high-affinity antibodies. So, new kinds of kinase assays such as fluorescent, electrochemical, surface plasmon resonance, and mass spectroscopy have been developed. Although these methods have some advantages over traditionally assays, but they always involve in multiple steps such as labeling, incubation, washing, testing and so on. Therefore, developing highly efficient and general assays to detect protein kinase is still a challenge for the analyst.According to the above considerations, we took advantage of the high separation performance as well as automation of capillary electrophoresis method and the excellent enrichment capability of on-line sample preconcentration technique, thus achieved highly sensitive detection of protein kinase activity and high-throughput screening of kinase inhibitors. The primary coverages are summarized as follow:1. A novel method for on-line probing the protein kinase activity based on capillary isotachophoresis (CITP) by using discontinuous solution system is developed. CITP is not only capable of improving analytical performance and resolution, but can also be used as an on-line preconcentration technique to enhance the sensitivity of capillary electrophoresis. Due to the low concentration of phosphorylated product, a low limit of detection down to 12.5 mU/?L was achieved, thus greatly improve the detection sensitivity for protein kinase assays or screening of kinase inhibitors.2. An highly sensitive method for PKA activity detection based on on-line CIEF-LIF is presented. Here, an highly sensitive analysis of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase (PKA) activity was achieved in on-line CIEF-LIF analysis with detection sensitivity down to 1.25 mU/?L.3. Based on the high detection sensitivity of on-line CIEF, we realized the multiple kinases simultaneous analysis and high-throughput screening of its specific inhibitors in real cell lysates samples. The inhibition effect of inhibitors for corresponding kinases was estimated, with IC50 of H-89 for PKA and Ro-3306 for CDK1 calculated as 37.0 nM and 35.9 nM respectively. Experiments show that on-line CIEF platform can not only be used for multiple protein kinase simultaneous analysis and inhibitors throughput screening, but also for the PKA activity analysis in the actual samples of cell lysates.