Analysis of Protein Target Interactions of Synthetic Mixtures by Affinity-LC/MS.
Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90). It was possible to analyze quantitatively the binding of the components of the mixture to the target independently from each other without any preceding process such as purification. This feature has wide implications in biological sciences as crude mixtures, either natural or synthetic, can be analyzed directly for their possible binding to a target. This method could lead to savings in costs and labor through shortening chemical research project development time.
1 School of Biological Sciences, Nanyang Technological University (NTU), Singapore.
Prachi
P
Singh
2 Department of Pharmacy, National University of Singapore (NUS), Singapore.
Kalaipriya
K
Madhaiyan
1 School of Biological Sciences, Nanyang Technological University (NTU), Singapore.
Minh-Dao
MD
Duong-Thi
2 Department of Pharmacy, National University of Singapore (NUS), Singapore.
Brian W
BW
Dymock
1 School of Biological Sciences, Nanyang Technological University (NTU), Singapore.
Sten
S
Ohlson
D016227:Benzoquinones / Q000737:chemistry; D001593:Benzyl Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D047029:Lactams, Macrocyclic / Q000737:chemistry; D011485:Protein Binding; D054852:Small Molecule Libraries / Q000737:chemistry*; D012996:Solutions; D053719:Tandem Mass Spectrometry / Q000379:methods
D016227:Benzoquinones; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D054852:Small Molecule Libraries; D012996:Solutions; D016650:Fluorescein-5-isothiocyanate; C001277:geldanamycin
Hsp90; WAC; affinity-LC/MS; crude mixture; inhibitors; molecular interaction