High performance liquid chromatography coupled on-line to capillary electrophoresis with laser-induced fluorescence detection for detecting inhibitors of Src homology 2 domain-phosphopeptide binding in mixtures.
Reversed-phase HPLC was coupled on-line to a rapid, competitive affinity probe capillary electrophoresis (APCE) assay to screen mixtures for compounds that inhibit protein-ligand interactions. The Fyn Src homology 2 (SH2) domain and its phosphopeptide binding partner were used as a model interaction for demonstration of this technique. In the method, mixtures containing possible inhibitors of binding were separated by HPLC at a flow rate of 0.3 mL/min. A small portion of effluent was directed to a fluidic tee where it was mixed on-line with Fyn SH2 domain and a fluorescent phosphopeptide ("affinity probe") known to bind selectively to Fyn SH2 domain. Electropherograms of the reaction mixture were collected on-line at approximately 6s intervals using a flow-gated interface to control injections onto the capillary electrophoresis with laser-induced fluorescence system. The resulting electropherograms contained two peaks, one corresponding to the free affinity probe and the other a complex of the affinity probe and Fyn SH2 domain. Compounds that bound the protein were detected as a decrease in the peak height of the complex and an increase in the peak height of affinity probe with relative standard deviations of <5%. The assay was shown to resolve multiple peptidergic inhibitors and selectively detect them within a complex mixture of peptides. Signals were dependent upon both concentration of active peptide and its potency in binding inhibition. Detection limits were in the range of 2-11 microM depending upon the peptide. Common organic solvents used in HPLC were shown to have minimal effect in the on-line measurement up to approximately 60% in the mobile phase.
Yang
Peilin
P
Department of Chemistry, University of Michigan, 930 N. University, Ann Arbor, MI 48109-1055, USA.
Kennedy
Robert T
RT
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000494:pharmacology; D010748:Phosphopeptides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D018909:src Homology Domains
D010455:Peptides; D010748:Phosphopeptides