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 timeof-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.