Data-independent acquisition (DIA) is a powerful mass spectrometric technique to perform both protein identification and quantification of complex protein samples. Setting up DIA methods on Orbitrap analyzers requires a thorough overview of the actions the Orbitrap mass spectrometers carry out. This Tutorial is written with the intention to give an overview of the important parameters to consider as well as which measurements to carry out to get the most out of your DIA method when setting it up on an Orbitrap mass analyzer. Instead of giving the optimal DIA settings, all steps in the construction and optimization of the DIA method are shown and discussed in a way that allows tailored DIA methods. They key steps are building the spectral library after sample fractionation, deciding upon the number of data points per chromatographic peak, determining the scan times of each mass spectrometric step, constructing various DIA methods using these data, and evaluating their performance. This proposed DIA method development strategy was tested on digested lysates from Pseudomonas aeruginosa and compared with conventional DDA analysis to put the DIA results into perspective.

中文翻译：

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Orbitrap Q Exactive HF的数据独立采集：教程。

数据独立采集（DIA）是一种功能强大的质谱技术，可进行蛋白质鉴定和复杂蛋白质样品的定量分析。在Orbitrap分析仪上设置DIA方法需要全面了解Orbitrap质谱仪执行的操作。编写本教程的目的是概述要考虑的重要参数，以及在Orbitrap质量分析仪上进行设置时要进行哪些测量以最大程度地利用DIA方法。代替给出最佳DIA设置，而是以允许定制DIA方法的方式显示和讨论DIA方法的构建和优化的所有步骤。他们的关键步骤是在样品分离后建立光谱库，确定每个色谱峰的数据点数量，确定每个质谱步骤的扫描时间，使用这些数据构建各种DIA方法，并评估其性能。在铜绿假单胞菌的消化液中测试了该拟议的DIA方法开发策略，并将其与常规DDA分析进行了比较，从而使DIA的结果成为现实。