ALIS An Affinity Selection Mass Spectrometry System based on Continuous SEC

The schematic shown in Fig. 3.1 describes an optimized, integrated SEC-RPC-MS-based affinity selection platform developed at NeoGenesis, dubbed the

Mass Spectrometry Pharmacology
Fig. 3.1 Schematic of ALIS, an automated ligand identification system that uses continuous size-exclusion chromatography for the study of protein-ligand interactions.

Automated Ligand Identification System, or ALIS [40]. The ALIS system incorporates the following components: (1) an affinity selection stage, where a protein target binds to ligands of moderate to high affinity (Kd values of 10 mM to sub-nanomolar); (2) an SEC step that separates the protein-ligand complexes from unbound compounds; (3) an RPC step that dissociates the ligands from the complex; and (4) identification and quantitation of the dissociated ligands by MS.

In the affinity selection step, a protein target is incubated with one or more compounds in a physiologically relevant buffer containing any necessary cofac-tors, salts, metal ions, and detergents. The ALIS system is generic with respect to target class, and the binding reaction can be performed using proteins of virtually any variety, including both soluble targets and membrane-associated proteins; enzymes such as proteases, kinases, and phosphatases; nuclear hormone receptors; and G protein-coupled receptors (GPCRs). Genomic targets from bacterial or viral pathogens, especially novel target proteins that are derived from lethal gene product deletions but are of otherwise unknown function, can be readily studied to yield potential anti-infective compounds. Since the binding reaction is solution based, potential ligands can query all protein surfaces and not just the ''active site,'' enabling the discovery of ligands that act through allosteric binding and other mechanisms. The use of high-sensitivity MS enables ALIS to be used to characterize protein-ligand interactions while consuming only microgram amounts of a purified, soluble protein target.

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  • alice
    How affinity selection mass spec works?
    2 months ago

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