Steady improvement in Orbitrap‐based mass spectrometry (MS) technologies has greatly advanced the peptide sequencing speed and depth. In‐depth analysis of the performance of state‐of‐the‐art MS and optimization of key parameters can improve sequencing efficiency. In this study, we first systematically compared the performance of two popular data‐dependent acquisition approaches, with Orbitrap as the first‐stage (MS1) mass analyzer and the same Orbitrap (high‐high approach) or ion trap (high‐low approach) as the second‐stage (MS2) mass analyzer, on the Orbitrap Fusion mass spectrometer. High‐high approach outperformed high‐low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. However, regardless of the acquisition method, there are still more than 60% of peptide features untargeted for MS2 scan. We then systematically optimized the MS parameters using the high‐high approach. Increasing the isolation window in the high‐high approach could facilitate faster scan speed, but decreased MS2 identification rate. On the contrary, increasing the injection time of MS2 scan could increase identification rate but decrease scan speed and the number of identified MS2 spectra. Dynamic exclusion time should be set properly according to the chromatography peak width. Furthermore, we found that the Orbitrap analyzer, rather than the analytical column, was easily saturated with higher loading amount, thus limited the dynamic range of MS1‐based quantification. By using optimized parameters, 10 000 proteins and 110 000 unique peptides were identified by using 20 h of effective liquid chromatography (LC) gradient time. The study therefore illustrated the importance of synchronizing LC‐MS precursor ion targeting, fragment ion detection, and chromatographic separation for high efficient data‐dependent proteomics.

中文翻译：

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Orbitrap Tribrid 质谱仪液相色谱和质谱参数的协同优化，用于高效的数据依赖蛋白质组学

基于 Orbitrap 的质谱 (MS) 技术的稳步改进极大地提高了肽测序的速度和深度。深入分析最先进的质谱性能并优化关键参数可以提高测序效率。在这项研究中，我们首先系统地比较了两种流行的数据相关采集方法的性能，其中 Orbitrap 作为第一级 (MS1) 质量分析器和相同的 Orbitrap（高-高方法）或离子阱（高-低方法）作为第二级 (MS2) 质量分析器，在 Orbitrap Fusion 质谱仪上。在更好的扫描周期饱和度和更高的 MS2 识别率方面，高-高方法优于高-低方法。然而，无论采用何种采集方法，仍有超过 60% 的肽段特征不是 MS2 扫描的目标。然后，我们使用高-高方法系统地优化了 MS 参数。在 high-high 方法中增加隔离窗口可以促进更快的扫描速度，但会降低 MS2 识别率。相反，增加 MS2 扫描的进样时间可以提高识别率，但会降低扫描速度和已识别 MS2 谱图的数量。动态排阻时间应根据色谱峰宽适当设置。此外，我们发现 Orbitrap 分析器，而不是分析柱，很容易因更高的加载量而饱和，从而限​​制了基于 MS1 的定量的动态范围。通过使用优化的参数，使用 20 小时的有效液相色谱 (LC) 梯度时间鉴定了 10 000 种蛋白质和 110 000 种独特的肽。.