On-line continuous-flow, multi-protein biochemical assays for the characterization of bioaffinity compounds using electrospray quadrupole time-of-flight mass spectrometry.
The applicability of a homogeneous on-line continuous-flow, multi-protein biochemical assay was demonstrated for the interaction between fluorescein-biotin and streptavidin and for digoxin and anti-digoxigenin using electrospray quadrupole time-of-flight mass spectrometry (Q-TOF MS). In the on-line continuous-flow biochemical MS-based system several receptors (e.g., streptavidin and anti-digoxigenin, respectively) were allowed to react with corresponding reporter ligands (e.g.,fluorescein-biotin and digoxin, respectively). The methodology presented allows the simultaneous measurement of affinity and molecular mass of an active compound. By using automated MS and MS-MS switching functions of the Q-TOF, structure information is obtained allowing the characterization of bioactive compounds. No cross-reactivities were observed between the two model systems fluorescein-biotin/streptavidin and digoxin/anti-digoxigenin.
Faculty of Sciences, Division of Chemistry, Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands.
R J E
RJ
Derks
A C
AC
Hogenboom
G
G
van der Zwan
H
H
Irth
D000465:Algorithms; D002021:Buffers; D020650:Combinatorial Chemistry Techniques; D004076:Digoxigenin / Q000032:analysis; D004077:Digoxin / Q000032:analysis; D008024:Ligands; D009862:Online Systems; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D002021:Buffers; D008024:Ligands; D011506:Proteins; D004077:Digoxin; D004076:Digoxigenin