title: Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-Bbeta as a model system.
abstract: Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Peilin
authors.initials: P
authors.lastname: Yang
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Whelan
authors.forename: Emily E
authors.initials: EE
authors.lastname: Jameson
authors.forename: Jason H
authors.initials: JH
authors.lastname: Kurzer
authors.forename: Lawrence S
authors.initials: LS
authors.lastname: Argetsinger
authors.forename: Christin
authors.initials: C
authors.lastname: Carter-Su
authors.forename: Abuzar
authors.initials: A
authors.lastname: Kabir
authors.forename: Abdul
authors.initials: A
authors.lastname: Malik
authors.forename: Robert T
authors.initials: RT
authors.lastname: Kennedy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001896:Boron Compounds / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D019793:Fluorescein / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006207:Half-Life; D053614:Janus Kinase 2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D048868:Adaptor Proteins, Signal Transducing; D001896:Boron Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C115327:Sh2bpsm1 protein, mouse; D014443:Tyrosine; C045132:4-carboxyfluorescein; D053614:Janus Kinase 2; D019793:Fluorescein