Phosphate‐affinity electrophoresis on a microchip for determination of protein kinase activity

Abstract

We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form. We synthesized a Phos-tag-poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS-glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos-tag-poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c-Src-reacted samples, the results from the microPAE were in good agreement with those from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The microPAE was also successful in the presence of inhibitors for c-Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values.

The Cutting Edge of Affinity Electrophoresis Technology

“…Han et al synthesized a linear copolymer of acrylamide-pendent Phos-tag and dimethylacrylamide as a separation carrier that they used to fill the channel of a microchip carrier by autonomous injection to form a microchip suitable for use in phosphate affinity electrophoresis [ 18 , 19 , 20 ]. This method is extremely useful for rapid quantitative analysis of the phosphorylation/dephosphorylation ratios of substrate peptides after in vitro kinase or phosphatase reactions.…”

“…In addition, affinity electrophoresis techniques based on the retardation of the migration of molecules that bind strongly to affinity probes immobilized on a supported matrix have also been developed, and these have contributed to analyses of major posttranslational modifications of proteins [ 11 , 12 , 13 , 14 ]. Various types of apparatus for affinity electrophoresis have been developed for use in such techniques as capillary electrophoresis [ 15 , 16 , 17 ] and microchip electrophoresis [ 18 , 19 , 20 ]. Capillary affinity electrophoresis, in particular, has been widely used as a technique for various modes in the field of life science [ 21 ].…”

Recent developments in CE and CEC of peptides (2009–2011)

“…Microchip phosphate‐ACE utilizing autonomous sample injection for PDMS microchips and synthetic phosphate‐specific affinity ligand makes possible to trap phosphorylated peptides and separate them within 10 s from non‐phosphorylated peptides 236. In the proof‐of‐concept experiments, the method showed great promise for fast determination of peptide and protein kinase activity in the microscale, up to a single‐cell level.…”

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

“…CE with inherent advantages such as high separation efficiency, convenient for operation, liable for automation, and throughput analysis, makes it an attractive method for enzyme assay and drug discovery . Nowadays, CE‐based assays have generated wide applications in kinase activity analysis and inhibitor throughput screening. However, unsatisfactory sensitivity is often encountered in CE assay due to its limited sample injection amount, resulted in enzyme or substrate of high concentration often required, thus unfavorable for real sample application and inhibitors efficient screening.…”