© 2005 The Japanese Biochemical Society.
Regular Paper |
Affinity-Tagged Phosphorylation Assay by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (ATPA-MALDI): Application to Calcium/Calmodulin-Dependent Protein Kinase
1 Human Stress Signal Research Center, 2 Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda 563-8577; and 3 Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA
* To whom correspondence should be addressed. Tel: +81-72-751-8242, Fax: +81-72-751-9964, E-mail: t.kinumi{at}aist.go.jp
A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)–based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.