A non-radioactive in vitro CaMKII activity assay using HPLC-MS

Introduction: Calcium/Calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase that phosphorylates and regulates activity of many substrates in various tissues. Traditional CaMKII activity assays rely on incorporation of radioactivity onto a CaMKII substrate by utilizing gamma-P-32 ATP, which has a short half-life and can pose health risks to the researchers. Methods: An 8-minute HPLC-MS method was developed to measure a CaMKII-specific peptide substrate autocamtide- 2 (AC-2) and its phosphorylated form, phosphoautocamtide-2 (PAC-2). Degradation of AC-2 and PAC-2 in solutions and how to stabilize them were studied. The method was validated according to FDA guidelines for bioassays, and applied to determine CaMKII activity in a C2C12 cell lysate and IC50 of KN-93, a known CaMKII inhibitor. Results: Simple acidification with formic acid prevented AC-2 and PAC-2 from undergoing rapid degradation in the CaMKII assay mixture and in diluted water solutions. LLOQ of the HPLC-MS method was 0.26 mu M and 0.12 mu M for quantification of AC-2 and PAC-2, respectively. Precision was within 15% and accuracy was within 100 +/- 15%. Using the developed method, IC50 of KN-93 was measured to be 399 +/- 66 nM, which was compatible to reported values. Conclusions: A validated HPLC-MS method provides precise and accurate determination of AC-2 and PAC-2. This method enabled enzyme activity assay and inhibitor IC50 determination for CaMKII without radioactive labelled reagents.