NONRADIOACTIVE PHOSPHOPEPTIDE ASSAY BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY - APPLICATION TO CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE-II/

Matrix-assisted laser desorption ionization time-of-flight mass spectr ometry (MALDI-TOF) was used to quantify the phosphopeptide produced by calcium/calmodulin-dependent protein kinase II (CaMK II). MALDI-TOF m easurements were performed in a linear and positive ion mode with dela yed extraction excited at various laser powers and at different sampli ng positions, i.e., different loci of laser illumination. We find that the ratio of the peak area of the substrate (S) to that of its monoph osphorylated form (SP) for a given mixture is constant, independent of the laser powers and/or of the sample loci illuminated by the laser. We also find that the fraction of phosphorylation determined by MALDI- TOF, or f(MALDI-TOF), is proportionally smaller than that determined b y HPLC, or f(HPLC); the ratio f(MALDI-TOF)/f(HPLC) was 0.797 +/- 0.022 9 (99% confidence limit, n = 7) for a 30-mer peptide substrate used in this study. A low mass gate, which turns off the detector temporarily , improved the ratio f(MALDI-TOF)/f(HPLC) to 0.917 +/- 0.0184 (99% con fidence limit, n = 7). Our interpretation of this result is that the r eduction of the phosphopeptide peak in the MALDI-TOF measurement is li kely to be caused by a temporal loss of detector function rather than by a lower efficiency of ionization for the phosphopeptide compared wi th its parent species. In these measurements the experimental errors, up to the 50% phosphorylation state, were less than 5%. After an adjus tment made based on the f(MALDI-TOF)/f(HPLC) ratio of 0.917, MALDI-TOF gave an accurate measurement for the kinetics of the CaMK II phosphor ylation reaction. Since only a small volume of the reaction mixture, t ypically containing 3 to 50 pmol of substrate, is required for the MAL DI-TOF measurement, this method can be adapted to a nonradioactive mic roscale assay for CaMK II and also for other protein kinases. (C) 1998 Academic Press.