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.