Capillary Electrophoresis and Fluorescence Anisotropy for Quantitative Analysis of Peptide-Protein Interactions Using JAK2 and SH2-B
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- 作者：Peilin Yang ; Rebecca J. Whelan ; Emily E. Jameson ; Jason H. Kurzer ; Lawrence S. Argetsinger ; Christin Carter-Su ; Abuzar Kabir ; Abdul Malik ; and Robert T. Kennedy
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：April 15, 2005
- 年：2005
- 卷：77
- 期：8
- 页码：2482 - 2489
- 全文大小：160K
- 年卷期：v.77,no.8(April 15, 2005)
- ISSN：1520-6882
文摘
Fluorescence anisotropy capillary electrophoresis (FACE)and affinity probe capillary electrophoresis (APCE) withlaser-induced fluorescence detection were evaluated foranalysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-B
(SH2-B (525-670)) and a tyrosine-phosphorylatedpeptide corresponding to the binding sequence of JAK2were used as a model system. For peptide labeled withfluorescein, the Kd = 82 ± 7 nM as measured byfluorescence anisotropy (FA). APCE assays had a limit ofdetection (LOD) of 100 nM or 12 amol injected for SH2-B (525-670). The separation time of 4 s, achieved usingan electric field of 2860 V/cm on 7-cm-long capillaries,was on the same time scale as complex dissociationallowing Kd (101 ± 12 nM in good agreement with FAmeasurements) and dissociation rate (koff = 0.95 ± 0.02s-1 corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higherrate of complex dissociation than what had previouslybeen measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detectedwith an LOD of 300 nM or 7.5 fmol injected. FACE wasnot used for determining Kd or koff; however, this methodprovided better separation resolution for multiple formsof the protein than APCE. Both methods were foundsuitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complementsto existing techniques for exploring binding interactionswith rapid kinetics.
(SH2-B (525-670)) and a tyrosine-phosphorylatedpeptide corresponding to the binding sequence of JAK2were used as a model system. For peptide labeled withfluorescein, the Kd = 82 ± 7 nM as measured byfluorescence anisotropy (FA). APCE assays had a limit ofdetection (LOD) of 100 nM or 12 amol injected for SH2-B (525-670). The separation time of 4 s, achieved usingan electric field of 2860 V/cm on 7-cm-long capillaries,was on the same time scale as complex dissociationallowing Kd (101 ± 12 nM in good agreement with FAmeasurements) and dissociation rate (koff = 0.95 ± 0.02s-1 corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higherrate of complex dissociation than what had previouslybeen measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detectedwith an LOD of 300 nM or 7.5 fmol injected. FACE wasnot used for determining Kd or koff; however, this methodprovided better separation resolution for multiple formsof the protein than APCE. Both methods were foundsuitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complementsto existing techniques for exploring binding interactionswith rapid kinetics.