Mass spectrometric methods for biomolecular screening

A method for identifying a binding site for a ligand on a biomolecular target comprising:

• (a) collecting mass spectral fragmentation data for said biomolecular target;
• (b) providing a complex of said biomolecular target and said binding agent;
• (c) ionizing said complex in a mass spectrometer to provide one or more ions of said complex;
• (d) fragmenting at least one of said ions deriving from the complex;
• (e) collecting fragmentation data from said fragmentation of the ions from the complex;
• and
• (f) relating the fragmentation data of the ions of the complex with the fragmentation data from the biomolecular target to determine said site of binding.

A method for identifying a compound which binds to a preselected biomolecular target, said compound being present in a mixture of compounds comprising:

• (a) providing a complex of said biomolecular target and a standard binding compound which binds to said target under conditions effective to achieve said binding;
• (b) combining with said complex under competitive binding conditions the mixture of compounds;
• (c) ionizing said combination in a mass spectrometer to provide a plurality of ions;
• (d) fragmenting at least one of said ions in a mass spectrometer;
• (e) collecting mass spectral data for the fragmentation;
• and
• (f) relating said mass spectral data to the existence and degree of competitive binding.

A method for identifying a compound which binds to a preselected biomolecular target, said compound being present in a mixture of compounds comprising:

• (a) providing a complex of said biomolecular target and a standard compound which binds to said target under conditions effective to achieve said binding;
• (b) acquiring fragmentation data from the mass spectrometric analysis of the complex;
• (c) combining with a further portion of said complex under competitive binding conditions the mixture of compounds;
• (d) ionizing said combination in a mass spectrometer to provide a plurality of ions;
• (e) fragmenting at least one of said ions in a mass spectrometer,
• (f) collecting mass spectral data for the fragmentation;
• and
• (g) relating the mass spectral data acquired in steps (b) and (f) to the existence and degree of competitive binding of said compound.

A method for identifying binding sites of a biomolecular target for compounds from a combinatorial library comprising:

• (a) providing mass spectral fragmentation data for said biomolecular target;
• (b) providing a first complex of said biomolecular target and a standard binding compound which binds to said target;
• (c) combining with said first complex a combinatorial mixture of compounds;
• (d) ionizing in a mass spectrometer said combination from step Ĉ to provide a plurality of ions for said combination;
• (e) fragmenting at least one of said ions in a mass spectrometer to generate fragmentation data;
• (f) relating the fragmentation data collected for said biomolecular target and the ionized combination from step (d) to afford said identification.

A method for determining the nature and extent of binding of a ligand with a molecular interaction site of a biomolecule comprising

• contacting the biomolecule with the ligand under conditions selected to promote binding of the ligand to the molecular interaction site to give rise to a complex of said biomolecule and the ligand;
• ionizing the complex in a mass spectrometer;
• fragmenting the ionized complex;
• and
• determining whether the ligand binds to the molecular interaction site and, if so, determining the strength of binding of such ligand as compared to other ligands bound to said molecular interaction site.

A method of identifying chemical ligands which bind with high specificity and affinity to a molecular interaction site of an RNA comprising

• preparing a library of ligands in accordance with a ranked hierarchy of such ligands predicted or calculated to be bindable to the molecular interaction site;
• contacting the RNA with the ligand library under conditions selected to promote binding of the ligand library to the molecular interaction site of the RNA to give rise to complexes of said RNA and the ligands of the library;
• ionizing the complexes in a mass spectrometer;
• fragmenting the ionized complexes;
• and
• determining whether the ligand of each such complex binds to the molecular interaction site of the RNA and, if so, determining the strength of binding of such ligand as compared to the binding strength of other ligands of the library.