Evaluation and optimization of mass spectrometric settings during data-dependent acquisition mode: focus on LTQ-Orbitrap mass analyzers.
Evaluation and optimization of mass spectrometric settings during data-dependent acquisition mode: focus on LTQ-Orbitrap mass analyzers.
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DOI：
10.1021/pr3011588
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发表时间：
2013-07-05
影响因子：
4.4
通讯作者：
Hess S
中科院分区：
文献类型：
--
作者：
Kalli A;Smith GT;Sweredoski MJ;Hess S
基于质谱的蛋白质组学已经发展成为分析复杂蛋白质组的首选方法。毫无疑问，质谱仪器的最新进展极大地增强了蛋白质组学分析。LTQ-Orbitrap质量分析仪是蛋白质组学研究中一种流行的仪器平台。在本教程中，我们将讨论在CID数据依赖采集（DDA）模式下评估和优化LTQ-Orbitrap上的质谱设置以提高蛋白质和肽识别率的重要性。我们专注于那些MS和MS/MS参数，这已经被一些研究人员系统地检查和评估，并在DDA期间常用。更具体地说，我们讨论了质量分辨能力的影响，预览模式FTMS扫描，单一同位素前体选择，触发MS/MS事件的信号阈值，每次MS/MS扫描的微扫描次数，MS/MS事件的次数，MS和MS/MS的自动增益控制目标值（离子总数），MS/MS的最大离子注入时间，快速和正常的扫描速率和离子注入时间的预测。此外，我们还提供了来自最新一代LTQ-Orbitrap系统（Orbitrap Elite）的数据，沿着推荐的MS和MS/MS参数。Orbitrap Elite在扫描速度、灵敏度、动态范围、分辨率方面优于Orbitrap Classic，从而实现更高的识别率。在LTQ-Orbitrap Classic和XL上确定的几个优化MS参数很容易转移到Orbitrap Elite上，而其他参数需要重新评估。最后，简要讨论了Q Exactive和HCD，以及样品制备，LC优化和生物信息学分析。我们希望本教程将作为新的蛋白质组学领域的研究人员的指导，并协助实现最佳结果。
Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters, which have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate and prediction of ion injection time. We, furthermore, present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, resolving power and resulting in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as, sample preparation, LC-optimization and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results.
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