Evaluation of Data-Dependent and -Independent Mass Spectrometric Workflows for Sensitive Quantification of Proteins and Phosphorylation Sites
Evaluation of Data-Dependent and -Independent Mass Spectrometric Workflows for Sensitive Quantification of Proteins and Phosphorylation Sites
复制标题
DOI：
10.1021/pr500860c
复制
发表时间：
2014-12-01
影响因子：
4.4
通讯作者：
Schmidt, Alexander
中科院分区：
文献类型：
--
作者：
Bauer, Manuel;Ahrne, Erik;Schmidt, Alexander
近年来，定向质谱工作流程（特别是定向质谱工作流程）作为传统数据依赖采集 (DDA) LC-MS/MS 方法的替代技术已获得发展动力。通过关注特定的肽种类，这些方法可以对选定的感兴趣的蛋白质进行假设驱动的分析，并且它们已被证明适合监测复杂混合物中的低丰度蛋白质。尽管它们越来越受欢迎，但还没有研究系统地评估这些不同的 MS 策略在应用于复杂样品时的定量、检测和识别极限。在这里，我们系统地比较了传统 DDA、定向和各种靶向 MS 方法在两种不同仪器（即混合线性离子阱 Orbitrap 和三重四极杆仪器）上的性能。我们通过分析 20 种未修饰的和 10 种磷酸化的合成重标记参考肽的稀释系列，分别评估了每种方法的鉴定、定量和检测的局限性，在有或没有复杂的人类细胞消化背景的情况下，肽浓度覆盖了 6 个数量级。我们发现，在没有背景蛋白质的情况下，所有方法的表现都相似；然而，在分析全细胞裂解物时，靶向方法的灵敏度至少比定向方法或 DDA 方法高 5-10 倍。特别是，使用线性离子阱对中性损失峰进行高级碎裂 (MS3)，将某些磷酸肽的动态定量范围提高了 100 倍。我们通过成功量化非富集下拉样本中不同细胞周期状态下着丝粒和纺锤体组装检查点组件 Mad1 的 9 个磷酸化位点，说明了这种靶向 MS3 方法用于磷酸肽监测的强大功能。
In recent years, directed and, particularly, targeted mass spectrometric workflows have gained momentum as alternative techniques to conventional data-dependent acquisition (DDA) LC-MS/MS approaches. By focusing on specific peptide species, these methods allow hypothesis-driven analysis of selected proteins of interest, and they have been shown to be suited to monitor low-abundance proteins within complex mixtures. Despite their growing popularity, no study has systematically evaluated these various MS strategies in terms of quantification, detection, and identification limits when they are applied to complex samples. Here, we systematically compared the performance of conventional DDA, directed, and various targeted MS approaches on two different instruments, namely, a hybrid linear ion trap-Orbitrap and a triple quadrupole instrument. We assessed the limits of identification, quantification, and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy-labeled reference peptides, respectively, covering 6 orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed similarly in the absence of background proteins; however, when analyzing whole-cell lysates, targeted methods were at least 5-10 times more sensitive than that of the directed or DDA method. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased the dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying nine phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from nonenriched pull-down samples.