Collision Energy Optimization of b- and y-Ions for Multiple Reaction Monitoring Mass Spectrometry
Collision Energy Optimization of b- and y-Ions for Multiple Reaction Monitoring Mass Spectrometry
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DOI：
10.1021/pr1004289
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发表时间：
2011-01-01
影响因子：
4.4
通讯作者：
Martin, Daniel B.
中科院分区：
文献类型：
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作者：
Holstein (Sherwood), Carly A.;Gafken, Philip R.;Martin, Daniel B.
多反应监测(MRM)是一种高度灵敏且日益流行的靶向质谱学(MS)方法，可用于选择性地检测和定量生物样品中感兴趣的多肽及其相应的蛋白质。MRM-MS的灵敏度高度依赖于过渡特定参数的调节，特别是在多肽碎裂过程中应用的碰撞能(CE)。目前，CE的经验公式对y型离子最有效，而对于其他类型的跃迁，如b型离子和特定酰胺键上的小y型跃迁，如果优化为最大信号传输，也可能对MRM-MS有用。在这项工作中，我们对80个双电荷肽的所有跃迁进行了CE优化，其结果被用来定义单独的CE方程，用于b-离子和y-离子，以及来自氨基末端天冬氨酸或谷氨酸残基结合的酰胺键(D/E-X跃迁)的小Y型离子的CE方程。这一分析产生了四个主要的观察结果：(1)对于最佳碎裂，b离子往往比y离子需要更低的碰撞能，而D/E-X跃迁往往需要更多的碰撞能量；(2)CE方程在考虑产物离子m/z相关性时更能准确地预测最佳CE；(3)对于y离子、b离子和D/E-X跃迁，单独的CE方程比以前的一刀切的方程更有效，但通过单独优化跃迁可以得到最好的结果；和(4)虽然b离子从CE优化中获得了实质性的信号-通常增加了几倍-但它们的排名仍然倾向于低于来自同一肽的y离子。这些结果证实了Y离子通常是MRM实验的首选跃迁的概念，但也首次证明，如果使用适当的碰撞能量，b离子也可以成为可行的靶子。
Multiple reaction monitoring (MRM) is a highly sensitive and increasingly popular method of targeted mass spectrometry (MS) that can be used to selectively detect and quantify peptides and their corresponding proteins of interest within biological samples. The sensitivity of MRM-MS is highly dependent upon the tuning of transition-specific parameters, especially the collision energy (CE) applied during peptide fragmentation. Currently, empirical equations for CE work best for y-type ions and are much less effective for other types of transitions, such as b-type ions and small y-type transitions across particular amide bonds, which could also be useful for MRM-MS if optimized for maximum signal transmission. In this work, we have performed a CE optimization of all transitions for 80 doubly charged peptides, the results of which were used to define separate CE equations for b-ions and y-ions, as well as for small y-type ions derived from the fragmentation of amide bonds bounded on the amino-terminal side by aspartic or glutamic acid residues (D/E-X transitions). This analysis yielded four major observations: (1) b-ions tend to require lower collision energies than y-ions for optimal fragmentation, while D/E-X transitions tend to require more; (2) CE equations predict the optimal CEs more closely when product ion m/z dependence is included, in addition to the current standard of precursor ion m/z dependence; (3) separate CE equations for y-ions, b-ions, and D/E-X transitions are more effective than the previous one-size-fits-all equations, but best results are achieved by optimizing transitions individually; and (4) while b-ions gain substantial signal from CE optimization-often increases of several-fold-they still tend to rank lower than y-ions from the same peptide. These results confirm the notion that y-ions are usually the first-choice transitions for MRM experiments but also demonstrate, for the first time, that b-ions can be viable targets as well, if the proper collision energies are used.