SCREENING FOR HIGH-AFFINITY LIGANDS TO THE SRC SH2 DOMAIN USING CAPILLARY ISOELECTRIC FOCUSING-ELECTROSPRAY IONIZATION ION-TRAP MASS-SPECTROMETRY

A solution-based microscale approach for determination of high-affinit y noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray i onization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentr ated up to 2 orders of magnitude and separated on the basis of their i soelectric points. The complexes are then dissociated in the mass spec trometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information f or the ligands involved in tight complex formation may be obtained usi ng the MSn capabilities of the ion trap. The methodology can potential ly be used to screen rapidly combinatorial mixtures of compounds for h igh-affinity ligands.