Online fluorescence enhancement assay for the acetylcholine binding protein with parallel mass spectrometric identification.
The acetylcholine binding protein (AChBP) is considered an analogue for the ligand-binding domain of neuronal nicotinic acetylcholine receptors (nAChRs). Its stability and solubility in aqueous buffer allowed the development of an online bioaffinity analysis system. For this, a tracer ligand which displays enhanced fluorescence in the binding pocket of AChBP was identified from a concise series of synthetic benzylidene anabaseines. Evaluation and optimization of the bioaffinity assay was performed in a convenient microplate reader format and subsequently transferred to the online format. The high reproducibility has the prospect of estimating the affinities of ligands from an in-house drug discovery library injected in one known concentration. Furthermore, the online bioaffinity analysis system could also be applied to mixture analysis by using gradient HPLC. This led to the possibility of affinity ranking of ligands in mixtures with parallel high-resolution mass spectrometry for compound identification.
BioMolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands. j.kool@few.vu.nl
Jeroen
J
Kool
Gerdien E
GE
de Kloe
Ben
B
Bruyneel
Jon S
JS
de Vlieger
Kim
K
Retra
Maikel
M
Wijtmans
Rene
R
van Elk
August B
AB
Smit
Rob
R
Leurs
Henk
H
Lingeman
Iwan J P
IJ
de Esch
Hubertus
H
Irth
D000691:Anabasine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid; D055808:Drug Discovery; D017022:Flow Injection Analysis; D005470:Fluorometry; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D011485:Protein Binding; D011869:Radioligand Assay; D015203:Reproducibility of Results
C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; D008024:Ligands; C091713:anabaseine; D000691:Anabasine