Parker Laurie, Engel-Hall Aaron, Drew Kevin, Steinhardt George, Helseth Donald L, Jabon David, McMurry Timothy, Angulo David S, Kron Stephen J
Ludwig Center for Metastasis Research, University of Chicago, Knapp R322, 924 E. 57th Street, Chicago, IL 60637, USA.
J Mass Spectrom. 2008 Apr;43(4):518-27. doi: 10.1002/jms.1342.
Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored.
尽管在使用质谱分析磷酸化肽的方法和仪器方面取得了进展，但由于未磷酸化肽和磷酸化肽之间的物理化学差异，仍然难以量化底物的磷酸化程度。在这里，我们报告了一系列实验，通过创建已知肽/磷酸化肽输入比率的校准曲线并分析其产生的信号强度比率，使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）来研究这些差异。这些校准曲线揭示了相对解吸/电离效率在序列依赖性差异方面的微妙之处，这是单点校准无法看到的。我们发现，观察到的磷酸化肽信号的行为具有可重复性，变化范围为5-10%。尽管这些数据使我们能够开始解决与模拟这些特性以及预测其他肽序列的相对信号强度相关的问题，但很明显，这种行为非常复杂，需要进一步探索。
