Document Detail


Online magnetic bead dynamic protein-affinity selection coupled to LC-MS for the screening of pharmacologically active compounds.
MedLine Citation:
PMID:  19476387     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.
Authors:
N Jonker; A Kretschmer; J Kool; A Fernandez; D Kloos; J G Krabbe; H Lingeman; H Irth
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Publication Detail:
Type:  Evaluation Studies; Journal Article    
Journal Detail:
Title:  Analytical chemistry     Volume:  81     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2009 Jun 
Date Detail:
Created Date:  2009-05-29     Completed Date:  2009-08-05     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4263-70     Citation Subset:  IM    
Affiliation:
BioMolecular Analysis Group, Department of Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
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MeSH Terms
Descriptor/Qualifier:
Chromatography, Liquid / methods
Cobalt / chemistry*
Equipment Design
Estrogen Receptor alpha / analysis,  chemistry,  metabolism
Histidine / chemistry
Humans
Ligands
Magnetics*
Mass Spectrometry / methods
Protein Binding
Proteins / analysis*,  chemistry,  metabolism*
Sensitivity and Specificity
Solid Phase Extraction / economics,  instrumentation*,  methods*
Chemical
Reg. No./Substance:
0/Estrogen Receptor alpha; 0/Ligands; 0/Proteins; 71-00-1/Histidine; 7440-48-4/Cobalt

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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