title: Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP.
abstract: A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.
authors.affiliation: Chemical Technologies, Biovitrum AB, Stockholm, Sweden. kurt.benkestock@biovitrum.com
authors.forename: Kurt
authors.initials: K
authors.lastname: Benkestock
authors.forename: Colleen K
authors.initials: CK
authors.lastname: Van Pelt
authors.forename: Tomas
authors.initials: T
authors.lastname: Akerud
authors.forename: Alistair
authors.initials: A
authors.lastname: Sterling
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Edlund
authors.forename: Johan
authors.initials: J
authors.lastname: Roeraade
mesh_terms: D017667:Adipocytes / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D019411:Clinical Laboratory Techniques; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008403:Mass Screening; D013058:Mass Spectrometry; D009206:Myocardium / Q000378:metabolism; D009363:Neoplasm Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature; D025521:Tumor Suppressor Proteins; D014867:Water / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008024:Ligands; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins; D014867:Water