Quantitative crosslinking mass spectrometry (QCLMS) reveals structural details of protein conformations in solution. QCLMS can benefit from data-independent acquisition (DIA), which maximises accuracy, reproducibility and throughput of the approach. This DIA-QCLMS protocol comprises of three main sections: sample preparation, spectral library generation and quantitation. The DIA-QCLMS workflow supports isotope-labelling as well as label-free quantitation strategies, uses xiSEARCH for crosslink identification, and xiDIA-Library to create a spectral library for a peptide-centric quantitative approach. We integrated Spectronaut, a leading quantitation software, to analyse DIA data. Spectronaut supports DIA-QCLMS data to quantify crosslinks. It can be used to reveal the structural dynamics of proteins and protein complexes, even against a complex background. In combination with photoactivatable crosslinkers (photo-DIA-QCLMS), the workflow can increase data density and better capture protein dynamics due to short reaction times. Additionally, this can reveal conformational changes caused by environmental influences that would otherwise affect crosslinking itself, such as changing pH conditions. SIGNIFICANCE: This protocol is an detailed step-by-step description on how to implement our previously published DIA-QCLMS workflow (Müller et al. Mol Cell Proteomics. 2019 Apr;18(4):786-795). It includes sample preparation for QCLMS, Optimization of DIA strategies, implementation of the Spectronaut software and required python scripts and guideline on how to analyse quantitative crosslinking data. The DIA-QCLMS workflow widen the scope for a range of new crosslinking applications and this step-by-step protocol enhances the accessibility to a broad scientific user base.

中文翻译：

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通过定量交联质谱和数据独立采集研究蛋白质结构动力学的协议。

定量交联质谱（QCLMS）揭示了溶液中蛋白质构象的结构细节。QCLMS可以受益于独立于数据的采集（DIA），该方法可以最大程度地提高方法的准确性，可重复性和通量。该DIA-QCLMS协议包括三个主要部分：样品制备，光谱库生成和定量。DIA-QCLMS工作流程支持同位素标记和无标记定量策略，使用xiSEARCH进行交联识别，使用xiDIA-Library创建以肽为中心的定量方法的光谱库。我们集成了领先的定量软件Spectronaut，以分析DIA数据。Spectronaut支持DIA-QCLMS数据来量化交联。它可以用来揭示蛋白质和蛋白质复合物的结构动力学，甚至在复杂的背景下。与可光活化的交联剂（photo-DIA-QCLMS）结合使用，由于反应时间短，工作流程可以提高数据密度并更好地捕获蛋白质动力学。此外，这可以揭示由环境影响引起的构象变化，否则会影响自身的交联，例如不断变化的pH条件。意义：该协议是有关如何实施我们先前发布的DIA-QCLMS工作流程的详细分步说明（Müller等人，Mol Cell Proteomics。2019 Apr; 18（4）：786-795）。它包括用于QCLMS的样品制备，DIA策略的优化，Spectronaut软件的实施以及所需的python脚本以及有关如何分析定量交联数据的指南。