Kinase Assay by Mass Spec

ANALYTICAL CHEMISTRY

Peptides immobilized on a surface plus mass spectrometry equals rapid and direct analysis of kinase activities, a new study shows [Angew. Chem. Int. Ed., 43, 5973 (2004)]. Developed by University of Chicago researchers Dal-Hee Min, Jing Su, and Milan Mrksich, the strategy avoids the complications of traditional kinase assays, including use of radioactive labels or antibody binding.

Kinases mediate the phosphorylation of specific substrates. They have many regulatory functions, and assaying kinase activity is key in many research areas, including drug discovery.

Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the Chicago team evaluates kinase activity from the spectra of peptides, immobilized on a gold substrate, before and after exposure to a kinase-mediated phosphorylation. In the after spectrum, the mass peak of the kinase's substrate is replaced by that of the product.

The team applied the method to six kinases. The kinase reaction yielded a mass change of 80, due to addition of a phosphate group to each substrate. Multiple kinase activities can be measured by exposing a peptide array sequentially to different kinases.

The assay also can be used to measure kinase inhibition. Results for the inhibition of the kinase Ab1 by the anticancer drug Gleevec are within the range of values reported in the literature.

"The most significant aspect of our work has been the development of surfaces that allow both quantitative assays of enzyme activity and readout by mass spectrometry," Mrksich says. "These tailored substrates simplify the use of mass spectrometry for analyzing enzymatic activities and therefore move this technique closer to routine use in research laboratories."