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

Title:
Multiplexed Detection of Protein-Peptide Interaction and Inhibition Using Capillary Electrophoresis
Authors:
Yang, Peilin; Whelan, Rebecca J. ( 0000-0002-9293-1528 ) ; Mao, Yingwei; Lee, Angel W.-M.; Carter-Su, Christin; Kennedy, Robert T.
Abstract:
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.
Citation:
Yang, P., R.J. Whelan, Y. Mao, et al. 2007. "Multiplexed Detection of Protein-Peptide Interaction and Inhibition Using Capillary Electrophoresis." Analytical Chemistry 79(4): 1690-1695.
Publisher:
American Chemical Society
DATE ISSUED:
2007
Department:
Chemistry
Type:
article
PUBLISHED VERSION:
10.1021/ac061936e
PERMANENT LINK:
http://hdl.handle.net/11282/310272

Full metadata record

DC FieldValue Language
dc.contributor.authorYang, Peilinen_US
dc.contributor.authorWhelan, Rebecca J.en_US
dc.contributor.authorMao, Yingweien_US
dc.contributor.authorLee, Angel W.-M.en_US
dc.contributor.authorCarter-Su, Christinen_US
dc.contributor.authorKennedy, Robert T.en_US
dc.date.accessioned2013-12-23T16:29:24Z-
dc.date.available2013-12-23T16:29:24Z-
dc.date.issued2007en
dc.identifier.citationYang, P., R.J. Whelan, Y. Mao, et al. 2007. "Multiplexed Detection of Protein-Peptide Interaction and Inhibition Using Capillary Electrophoresis." Analytical Chemistry 79(4): 1690-1695.en_US
dc.identifier.issn0003-2700en_US
dc.identifier.urihttp://hdl.handle.net/11282/310272-
dc.description.abstractHigh-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.en_US
dc.publisherAmerican Chemical Societyen_US
dc.identifier.doi10.1021/ac061936e-
dc.subject.departmentChemistryen_US
dc.titleMultiplexed Detection of Protein-Peptide Interaction and Inhibition Using Capillary Electrophoresisen_US
dc.typearticleen_US
dc.identifier.journalAnalytical Chemistryen_US
dc.identifier.volume79en_US
dc.identifier.issue4en_US
dc.identifier.startpage1690en_US
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