GPC Spin Column HPLCESI-MS Methods for Screening Drugs Noncovalently Bound to Proteins

Secondary drug screening methods are described for determining the relative degree of non-covalent binding between drug candidates and a protein of therapeutic interest by gel centrifugation chromatography using GPC spin columns for isolating the protein–drug complexes, under native conditions, and reversed-phase HPLC coupled with ESI-MS for highly resolved and sensitive detection of the drug in the complex, under denaturing conditions. The necessary control samples and limitations of this work are fully described. The GPC spin column HPLC–ESI-MS methodology for screening of drugs non-covalently bound to proteins is illustrated for the non-covalent binding of geldanamycin with Hsp90cat protein.