Abstract In quantitative proteomics the use of data-independent acquisition (DIA) methods is getting increasingly popular and is applied to a growing number of sample types ranging from peptidomics to the analysis of post-translational modifications. Several of these sample types have been previously shown to profit from electron-based fragmentation methods such as ETD or EThcD. These fragmentation methods have so far not been implemented in DIA analysis. Here, we show the feasibility of combining DIA with EThcD fragmentation and provide insights into its performance. We show how EThcD can be used to increase peptide coverage, reaching similar success rates during targeted data extraction as standard HCD-based DIA. Furthermore, we illustrate how robust MS1 and LC results can be exploited to circumvent detrimental effects of less efficient fragmentation during targeted data extraction. Ultimately, our data demonstrates how EThcD-based MS/MS spectra can be connected to standard HCD-based DIA analyses, enabling the future use of decision-tree based spectral libraries to query large DIA data sets.

中文翻译：

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电子转移/高能碰撞解离 (EThcD) 对通过数据独立采集进行的磷酸肽分析的影响

摘要 在定量蛋白质组学中，数据独立采集 (DIA) 方法的使用越来越流行，并应用于越来越多的样本类型，从肽组学到翻译后修饰的分析。这些样品类型中的一些以前已被证明可以从 ETD 或 EThcD 等基于电子的碎裂方法中获益。迄今为止，这些碎片方法尚未在 DIA 分析中实施。在这里，我们展示了将 DIA 与 ETHcD 碎片相结合的可行性，并提供了对其性能的见解。我们展示了如何使用 EThcD 来增加肽覆盖率，在目标数据提取过程中达到与基于 HCD 的标准 DIA 相似的成功率。此外，我们说明了如何利用稳健的 MS1 和 LC 结果来规避目标数据提取期间效率较低的碎片的不利影响。最终，我们的数据展示了基于 EThcD 的 MS/MS 谱图如何连接到标准的基于 HCD 的 DIA 分析，从而使未来能够使用基于决策树的谱库来查询大型 DIA 数据集。