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Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP
KTH, Superseded Departments, Chemistry.
Advion BioSci Inc, Ithaca, NY USA.
Lund Univ, Dept Biophys Chem.
Advion BioSci Ltd, Norwich, Norfolk England.
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2003 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 8, no 3, 247-256 p.Article in journal (Refereed) Published
Abstract [en]

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. Furthermore, 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.

Place, publisher, year, edition, pages
2003. Vol. 8, no 3, 247-256 p.
Keyword [en]
automated nano-electrospray; noncovalent; screening; FABP; ACID-BINDING PROTEINS; COMPLEXES; MICRODIALYSIS; RESOLUTION; ADIPOCYTE; RECEPTOR
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-8358DOI: 10.1177/1087057103008003002ISI: 000183364000002OAI: oai:DiVA.org:kth-8358DiVA: diva2:13659
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-07-05Bibliographically approved
In thesis
1. Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery
Open this publication in new window or tab >>Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process.

The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available.

In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed.

The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. viii, 60 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2008:33
Keyword
Mass spectrometry, electrospray ionization, drug discovery, noncovalent interaction, complexes, human serum albumin (HSA), fatty acid binding protein (FABP), ribonuclease, ligand screening
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-4730 (URN)978-91-7178-947-1 (ISBN)
Public defence
2008-05-23, E2, KTH, Lindstedtsvägen 3, Stockholm, 00:00
Opponent
Supervisors
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-07-05Bibliographically approved

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