Stingl Christoph, Ihling Christian, Ammerer Gustav, Sinz Andrea, Mechtler Karl
Institute of Molecular Biotechnology (IMBA), Vienna, Dr.-Bohr-Gasse 3, 1030 Vienna, Austria.
Biochim Biophys Acta. 2006 Dec;1764(12):1842-52. doi: 10.1016/j.bbapap.2006.09.016. Epub 2006 Oct 3.
Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins, whereas ECD has proven especially valuable for mapping labile posttranslational modifications (PTMs), such as phosphorylations. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize phosphorylated peptides. For optimizing fragmentation parameters, sets of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation were analyzed. The influence of spectrum averaging for obtaining high-quality spectra was investigated. Our results show that the fragmentation methods CAD and ECD allow for a facilitated analysis of phosphopeptides; however, their general applicability for analyzing phosphopeptides has to be evaluated in each specific case with respect to the given analytical task. The major advantage of complementary peptide cleavages by combining different fragmentation methods is the increased amount of information that is obtained during MS/MS analysis of modified peptides. On the basis of the obtained results, we are planning to design LC time-scale compatible, data-dependent MS/MS methods using the different fragmentation techniques in order to improve the identification and characterization of phosphopeptides.
除了常用的碰撞诱导解离（CAD）外，电子捕获解离（ECD）和红外多光子解离（IRMPD）是傅里叶变换离子回旋共振质谱（FTICR-MS）中用于肽和蛋白质碎片化的互补技术。IRMPD和ECD都已被证明适用于肽和蛋白质的高效测序，而ECD已被证明对于绘制不稳定的翻译后修饰（PTM），如磷酸化图谱特别有价值。在这项工作中，我们比较了线性离子阱和ICR池中不同的碎片化技术和质谱检测，以评估它们有效鉴定和表征磷酸化肽的能力。为了优化碎片化参数，我们分析了一系列分子量约为1至4 kDa且磷酸化水平不同的合成肽。研究了光谱平均化对获得高质量光谱的影响。我们的结果表明，CAD和ECD碎片化方法有助于磷酸化肽的分析；然而，它们在分析磷酸化肽方面的普遍适用性必须根据给定的分析任务在每个具体案例中进行评估。通过结合不同的碎片化方法进行互补肽切割的主要优点是在修饰肽的串联质谱分析中获得的信息量增加。基于获得的结果，我们计划设计与液相色谱时间尺度兼容、数据依赖的串联质谱方法，使用不同的碎片化技术，以改进磷酸化肽的鉴定和表征。
