Multiplexed Detection of Protein−Peptide Interaction and Inhibition Using Capillary Electrophoresis

High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein−phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2007-02, Vol.79 (4), p.1690-1695
Main Authors: Yang, Peilin, Whelan, Rebecca J, Mao, Yingwei, Lee, Angel W.-M, Carter-Su, Christin, Kennedy, Robert T
Format: Article
Language:English
Subjects:
Analytical chemistry
Anisotropy
Binding sites
Biochemistry
Chemistry
Chromatographic methods and physical methods associated with chromatography
Electrophoresis, Capillary - instrumentation
Electrophoresis, Capillary - methods
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
Exact sciences and technology
Fluorescence
Molecular Structure
Molecular weight
Other chromatographic methods
Peptides
Peptides - analysis
Peptides - antagonists & inhibitors
Protein Binding
Proteins
Proteins - analysis
Proteins - antagonists & inhibitors
Sensitivity and Specificity
Spectrometric and optical methods
Structure-Activity Relationship
Time Factors
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Summary:High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein−phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins:  Src, SH2-Bβ, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-Bβ and Fyn complex barely affected. IC50 of both selective and nonselective inhibitors were determined and compared for different proteins. The IC50 of the nonselective inhibitor was 49 ± 9, 323 ± 42, and 228 ± 19 μM (n = 3) for Src, SH2-Bβ, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein−protein interactions.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac061936e