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

• Published in: Analytical chemistry (Washington) Vol. 79; no. 4; pp. 1690 - 1695
• Main Authors: Yang, Peilin, Whelan, Rebecca J, Mao, Yingwei, Lee, Angel W.-M, Carter-Su, Christin, Kennedy, Robert T
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.02.2007
• 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; Drug; Chemical analysis; Peptides; Capillary electrophoresis; High speed; Inhibitor; Selectivity; Inhibition; Molecular mass; Protein; Fluorescence spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac061936e

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.