Matsumoto H, Kahn E S, Komori N
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.
Anal Biochem. 1998 Jul 1;260(2):188-94. doi: 10.1006/abio.1998.2691.
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.
基质辅助激光解吸电离飞行时间质谱（MALDI - TOF）用于定量钙/钙调蛋白依赖性蛋白激酶II（CaMK II）产生的磷酸肽。MALDI - TOF测量是在线性正离子模式下进行的，采用延迟提取，在不同激光功率和不同采样位置（即激光照射的不同位点）激发。我们发现，对于给定的混合物，底物（S）与其单磷酸化形式（SP）的峰面积比是恒定的，与激光功率和/或激光照射的样品位点无关。我们还发现，通过MALDI - TOF测定的磷酸化分数，即fMALDI - TOF，比通过HPLC测定的分数，即fHPLC，成比例地小；对于本研究中使用的30聚体肽底物，fMALDI - TOF/fHPLC的比值为0.797±0.0229（99%置信限，n = 7）。一个低质量门控装置，它会暂时关闭检测器，将fMALDI - TOF/fHPLC的比值提高到了0.917±0.0184（99%置信限，n = 7）。我们对这一结果的解释是，MALDI - TOF测量中磷酸肽峰的降低可能是由检测器功能的暂时丧失引起的，而不是因为磷酸肽与其母体物种相比电离效率较低。在这些测量中，高达50%磷酸化状态的实验误差小于5%。基于0.917的fMALDI - TOF/fHPLC比值进行调整后，MALDI - TOF对CaMK II磷酸化反应的动力学进行了准确测量。由于MALDI - TOF测量仅需要少量的反应混合物，通常含有3至50皮摩尔的底物，因此该方法可适用于CaMK II以及其他蛋白激酶的非放射性微量分析。
