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On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies
KTH, Superseded Departments, Chemistry.
Biovitrum AB, Analyt Sci.
KTH, Superseded Departments, Chemistry.
2002 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 21, 2054-2059 p.Article in journal (Refereed) Published
Abstract [en]

Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The noncovalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS.

Place, publisher, year, edition, pages
2002. Vol. 16, no 21, 2054-2059 p.
Keyword [en]
IN-VIVO MICRODIALYSIS; NONCOVALENT COMPLEXES; LIQUID-CHROMATOGRAPHY; BIOLOGICAL SAMPLES; ESI-MS; PROTEIN; ONLINE; AFFINITY; IDENTIFICATION; INSTRUMENT
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-8359DOI: 10.1002/rcm.832ISI: 000178955900008OAI: oai:DiVA.org:kth-8359DiVA: diva2:13660
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2017-12-14Bibliographically 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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