Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
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 [en]
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: urn:nbn:se:kth:diva-4730ISBN: 978-91-7178-947-1 (print)OAI: oai:DiVA.org:kth-4730DiVA: diva2:13662
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
List of papers
1. Influence of droplet size, capillary-cone distance and selected instrumental parameters for the analysis of noncovalent protein-ligand complexes by nano-electrospray ionization mass spectrometry
Open this publication in new window or tab >>Influence of droplet size, capillary-cone distance and selected instrumental parameters for the analysis of noncovalent protein-ligand complexes by nano-electrospray ionization mass spectrometry
2004 (English)In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 39, no 9, 1059-1067 p.Article in journal (Refereed) Published
Abstract [en]

It has been suggested in the literature that nano-electrospray ionization (nano-ESI) mass spectrometry better reflects the equilibrium between complex and free protein in solution than pneumatically assisted electrospray ionization (ESI) in noncovalent interaction studies. However, no systematic studies of the effects of ionization conditions have been performed to support this statement. In the present work, different instrumental and sample-derived parameters affecting the stability of noncovalent complexes during analysis by nano-ESI were investigated. In general, increased values of parameters such as drying gas flow-rate, ion-source temperature, capillary tip voltage and buffer concentration lead to a dissociation of ribonuclease A (RNAse)-cytidine 2'-monophosphate (CMP) and cytidine 5'-triphosphate (CTP) complexes. The size of the electrosprayed droplets was shown to be an important issue. Increasing the capillary to cone distance yielded an increased complex to free protein ratio when a hydrophilic ligand was present and the reverse effect was obtained with a hydrophobic ligand. Important in this regard is the degree of sampling of ions originating from late-generation residue droplets, that is, ions present in the droplet bulk. Sampling of these ions increases with longer capillary-cone distance (flight time). Furthermore, when the sample flow-rate was increased by increasing the capillary internal tip i.d. from 4 to 30 mum, a decreased complex to free protein ratio for the RNAse-CTP system was observed. This behavior was consistent with the change in surface to volume ratio for flow-rates between 2 and 100 nl min(-1). Finally, polarity switching between positive and negative ion modes gave a higher complex to free protein ratio when the ligand and the protein had the same polarity.

Keyword
nano-electrospray ionization; noncovalent complexes; droplet size; capillary-cone distance; H-FABP; RNAse; BINDING INTERACTIONS; QUANTITATIVE-DETERMINATION; COMPETITIVE-BINDING; RESOLUTION; RECEPTOR; CHARGE; DNA
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-8357 (URN)10.1002/jms.685 (DOI)000223868000010 ()2-s2.0-4544221115 (Scopus ID)
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2011-09-23Bibliographically approved
2. Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP
Open this publication in new window or tab >>Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP
Show others...
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.

Keyword
automated nano-electrospray; noncovalent; screening; FABP; ACID-BINDING PROTEINS; COMPLEXES; MICRODIALYSIS; RESOLUTION; ADIPOCYTE; RECEPTOR
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-8358 (URN)10.1177/1087057103008003002 (DOI)000183364000002 ()
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-07-05Bibliographically approved
3. On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies
Open this publication in new window or tab >>On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies
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.

Keyword
IN-VIVO MICRODIALYSIS; NONCOVALENT COMPLEXES; LIQUID-CHROMATOGRAPHY; BIOLOGICAL SAMPLES; ESI-MS; PROTEIN; ONLINE; AFFINITY; IDENTIFICATION; INSTRUMENT
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-8359 (URN)10.1002/rcm.832 (DOI)000178955900008 ()
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-07-05Bibliographically approved
4. Electrospray ionization mass spectrometry as a tool for determination of drug binding sites to human serum albumin by noncovalent interaction
Open this publication in new window or tab >>Electrospray ionization mass spectrometry as a tool for determination of drug binding sites to human serum albumin by noncovalent interaction
2005 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 12, 1637-1643 p.Article in journal (Refereed) Published
Abstract [en]

Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work 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. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of 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. The advantages of nanoE-SI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.

Keyword
PROTEIN-LIGAND COMPLEXES; ULTRAFILTRATION; COMPETITION; RESOLUTION; AFFINITY; ACID
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-8360 (URN)10.1002/rcm.1967 (DOI)000229846800008 ()2-s2.0-20644463474 (Scopus ID)
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-07-05Bibliographically approved

Open Access in DiVA

fulltext(1083 kB)2788 downloads
File information
File name FULLTEXT01.pdfFile size 1083 kBChecksum MD5
43ed178ea482e9f62b715160e5317cbb48eeeee1f201afd1e81fd46cda4e39e78f88a75f
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Benkestock, Kurt
By organisation
Analytical Chemistry
Analytical Chemistry

Search outside of DiVA

GoogleGoogle Scholar
Total: 2788 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 820 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf