Isolation and Identification of Protein-Binding Ligands Using Bio-Solid-Phase Extraction/Tandem Mass Spectrometry

摘要

1. BioSPE has been demonstrated using various different protein systems including dihydrofolate reductase, anti-fluorescein and adenosine deaminase. 2. Extraction can be performed in an active-site specific manner using substrate, or using an protein denaturant such as an organic solvent. 3. Column reproducibility has been low thus there is a need to improve protein retention and prevent flow channeling of the mobile phase through the stationary phase. 4. Data dependent methodology has been developed to detect an unknown analyte eluting from a protein-doped column. 5. Detection limits of the data dependent assay needs to be improved; presently these improvements are being tested with the QTrap LIT that has sensitivity easily down to 50 nM analyte concentration, pre-concentration of the analyte on the column must be increased to this level at a minimum. 6. Matrix effects of eluting solvents needs to be assessed for data dependent method.