Bio-Solid-Phase Extraction and Information Dependent Tandem Mass Spectrometry for the Identification of Inhibitors in Complex Mixtures

摘要

BioSPE has been demonstrated using various different protein systems including dihydrofolate reductase, anti-fluorescein and adenosine deaminase. Extraction using 50% methanol/H_2O produces the most resolved peak but acetic acid is less harsh allowing for multiple uses on one columns. Extraction using substrate shows concentration dependence on inhibitor peak area. K_d is less potent, likely due to non-equilibrium conditions and partitioning effects. Column materials have been greatly improved by controlling the condensation time prior to mixing with the aqueous phase (backpressure # 500 psi at 5 μL/min). Data dependent methodology has been developed to detect an unknown analyte in a mixture but need a more sensitive system for sub-nM analysis → QTrap 2000 LIT and then OrbiTrap! Matrix effects of eluting solvents needs to be assessed for data dependent method. Increased mixture complexity must also be assessed → natural product extracts!