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 spectrometry (MALDI-TOF) was used to quantify the phosphopeptide produced by calcium/calmodulin-dependent protein kinase II (CaMK II). MALDI-TOF measurements were performed in a linear and positive ion mode with delayed extraction excited at various laser powers and at different sampling 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 monophosphorylated 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 fMALDI-TOF, is proportionally smaller than that determined by HPLC, or fHPLC; the ratio fMALDI-TOF/fHPLC was 0.797 +/- 0.0229 (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 fMALDI-TOF/fHPLC to 0.917 +/- 0.0184 (99% confidence limit, n = 7). Our interpretation of this result is that the reduction of the phosphopeptide peak in the MALDI-TOF measurement is likely to be caused by a temporal loss of detector function rather than by a lower efficiency of ionization for the phosphopeptide compared with its parent species. In these measurements the experimental errors, up to the 50% phosphorylation state, were less than 5%. After an adjustment made based on the fMALDI-TOF/fHPLC ratio of 0.917, MALDI-TOF gave an accurate measurement for the kinetics of the CaMK II phosphorylation reaction. Since only a small volume of the reaction mixture, typically containing 3 to 50 pmol of substrate, is required for the MALDI-TOF measurement, this method can be adapted to a nonradioactive microscale assay for CaMK II and also for other protein kinases.
Matsumoto
H
H
Department of Biochemistry and Molecular Biology and The NSF EPSCoR Oklahoma Laser Mass Spectrometry Facility, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Kahn
E S
ES
Komori
N
N
D000595:Amino Acid Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D010748:Phosphopeptides; D011994:Recombinant Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases