Screening Dyrk1A inhibitors by MALDI-QqQ mass spectrometry: systematic comparison to established radiometric, luminescence, and LC-UV-MS assays

摘要

Enzyme-catalyzed reactions play key roles in disease pathology, thus making them relevant subjects of therapeutic inhibitor screening experiments. Matrix-assisted laser desorption/ionization (MALDI) assays have been demonstrated to be able to replace established screening approaches. They offer increased sample throughput, but care must be taken to avoid instrumental bias from differences in ionization efficiencies. We compared a MALDI-triple-quadrupole(QqQ) method for the Dyrk1A peptide substrate woodtide to LC-MS, liquid chromatography with ultraviolet detection(LC-UV), luminescence, and radiometric assays. MALDI measurements were performed on a MALDI-QqQ instrument in the multiple-reaction monitoring mode. Different MALDI conditions were investigated to address whether matrix type, sample support, and MRM- or SIM-based detection conditions can be used to accommodate the molar responses of substrate peptide and its phosphorylated form. UV detection served as a reference method. The impact of MALDI matrix on IC_(50) values was small, even considering that matrix preparations were used that are known to alleviate response differences. IC(50) values determined by MALDI were ca. 2-fold lower than those determined by LC-UV. Although MALDI generated lower ion yields for the phosphorylated peptide than for the peptide substrate, we found that a correction of compound potencies was readily possible using correction factors based on unbiased LC-UVresults. A thorough method development delivered a robust assay with excellent performance(Z' > 0.91) that was close to that seen for LC-UV.