Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving “native” non‐covalent inter‐ and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high‐quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30–800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE‐MS setup. This study is aimed to help normalize nCZE‐MS practices to enhance the CE community and provide a resource for the production of reproducible and high‐quality data.

中文翻译：

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高达 800 kDa 的蛋白质和蛋白质复合物的天然 CZE-MS 标准程序

天然质谱 (nMS) 是一种快速发展的方法，用于表征大蛋白质和蛋白质复合物，保留“天然”非共价分子间和分子内相互作用。将纯化的分析物直接注入质谱仪代表了进行 nMS 实验的标准方法。或者，CZE 可以在天然条件下进行，提供高分离性能，同时消耗痕量的样品材料。在这里，我们提供了标准操作程序，用于使用 CZE 在本机模式下通过商业无鞘接口在线耦合到各种 Orbitrap 质谱仪获取高质量数据，涵盖 30-800 kDa 的各种分析物。使用标准蛋白质混合物，评估各种 CZE 方法参数的影响，例如 BGE/导电液体成分和分离电压。此外，针对模型分析物详细讨论了在蛋白质亚基和金属酶表征的背景下优化碎片设置的通用方法。一小部分专门介绍 nCZE-MS 设置的故障排除。本研究旨在帮助规范 nCZE-MS 实践，以增强 CE 社区并为生产可重复的高质量数据提供资源。