title: High-resolution bioactivity profiling of mixtures toward the acetylcholine binding protein using a nanofractionation spotter technology.
abstract: This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC(50)s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantage of this "atline" technology using postcolumn bioaffinity analysis when compared to continuous flow online postcolumn bioaffinity profiling is the possibility to choose postcolumn incubation times freely without compromising resolution due to diffusion effects.
authors.affiliation: BioMolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, Amsterdam, the Netherlands. j.kool@few.vu.nl
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
authors.forename: Ferry
authors.initials: F
authors.lastname: Heus
authors.forename: Gerdien
authors.initials: G
authors.lastname: de Kloe
authors.forename: Henk
authors.initials: H
authors.lastname: Lingeman
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.forename: Ewald
authors.initials: E
authors.lastname: Edink
authors.forename: Iwan J P
authors.initials: IJ
authors.lastname: De Esch
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
authors.forename: Wilfried M A
authors.initials: WM
authors.lastname: Niessen
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000032:analysis / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D005591:Chemical Fractionation / Q000379:methods; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045424:Complex Mixtures / Q000032:analysis* / Q000737:chemistry; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods*; D057166:High-Throughput Screening Assays; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D036103:Nanotechnology / Q000379:methods; D009538:Nicotine / Q000032:analysis / Q000378:metabolism; D018722:Nicotinic Agonists / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D011725:Pyridines / Q000032:analysis / Q000378:metabolism; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: C428944:AChBP protein, Lymnaea; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002352:Carrier Proteins; D045424:Complex Mixtures; D008024:Ligands; D018722:Nicotinic Agonists; D011725:Pyridines; D011994:Recombinant Proteins; D009538:Nicotine; C082748:epibatidine