| Abstract |
We describe a MALDI-TOF mass-spectrometry-based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALSG), CaMKII substrate (KRQQSFDLF), erktide (ATGPLSPGPFGRR), abltide (EAIYAAPFAKKK), srctide (AEEEIYGEFEAKKKK), neurogranin (AAAKIQASFRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMSITA). There are two fundamental points on which the proposed method is based to improve the mass-spectrometric response: 1) mass tag technology by N-derivatization through stable isotope labeling and 2) C-terminal conjugation with tryptophanylarginine (WR). It was suggested that C-terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5-13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac-EAIYAAPFAKKKWR-NH(2)), that WR conjugation at the C-terminus in combination with stable-isotope labeling at the N-terminus allowed the quantitative assay of recombinant c-Abl kinase in the presence of adenosine 5'-triphosphate (ATP; K(M,ATP)=18.6 microM and V(max)=642 pmol min(-1) microg(-1)). The present protocol made a simple and reliable inhibition assay of recombinant c-Abl kinase by imatinib possible (IC(50(recombinant))=291 nM; STI571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c-Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC(50(active form))=1049 nM and IC(50(inactive form))=54 nM). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer-cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple-kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high-throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope-free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.
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