Iterative size-exclusion chromatography coupled with liquid chromatographic mass spectrometry to enrich and identify tight-binding ligands from complex mixtures
1999
Abstract A method is described for the enrichment of tight-binding ligands from complex mixtures. The mixture is equilibrated with a protein or receptor, and protein: ligand complexes are separated from unbound ligands by size-exclusion. If the resulting mixture is allowed to re-establish its equilibrium and then passed again through the size-exclusion column, significant enrichment of tight-binding ligands is realized, even in cases where the weak-binders are present in large excess. Ligands are identified by liquid chromatography and mass spectrometry following the size-exclusion step. The process can be repeated multiple times to further enrich ligands. Analysis of pools of ligands generated by combinatorial chemistry is an obvious application of the technique.
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