Self-packed size-exclusion columns enable versatile high-throughput native, top-down, and ion-mobility-mass spectrometry studies on proteins and complexes.

Native MS (nMS) is a key structural biology technique that makes it possible to study intact proteins and their interactions. Unfortunately, non-volatile salts are incompatiblewith nMS, which demands a laborious desalting procedure. Non-denaturing size exclusion chromatography (SEC) allows both rapid desalting and separation and has previously been explored for nMS automation. However, SEC at conventional scale requires rather large sample amounts as well as harsh ESI conditions, which can cause protein unfolding. Capillary LC allows softer conditions; however, the few commercially available SEC columns appropriate for this flow rate are prohibitively expensive for many laboratories. Existing protocols for packing buffer exchange columns rely on specialized equipment and/or result in columns that have limited capacity for size-based protein separation. Here, we present self-packed miniaturized SEC columns with different stationary phases and customizable dimensions. The columns, produced via slurry packing with an ordinary LC pump were used across a range of samples in several applications including nMS, top-down MS (TDMS), ligand screening, and ion mobility (IM)-MS. Native separation allowed acquisition of data from samples containing more than one protein. We acquired native TDMS data of 3 proteins in 12 minutes, with up to 47% sequence coverage. IM-MS of alpha-synuclein at different charge states was measured in ca. 60 minutes (including calibrants), with results that match the literature. Finally, we used SEC-nMS to rapidly screen proteolysis-targeting chimera candidates and performed collision-induced unfolding (CIU) of a PROTAC-induced ternary complex. Through this work, we highlight the potential of SEC to support developments in structural MS.

天然质谱（Native MS，nMS）是一项关键的结构生物学技术，可用于研究完整蛋白质及其相互作用。遗憾的是，非挥发性盐与天然质谱不兼容，这需要耗时费力的脱盐流程。非变性尺寸排阻色谱（Non-denaturing size exclusion chromatography，SEC）可同时实现快速脱盐与分离，此前已有研究将其用于天然质谱的自动化操作。然而，常规规模的尺寸排阻色谱需要较大的样品用量，且需使用严苛的电喷雾电离（Electrospray Ionization，ESI）条件，这可能导致蛋白质变性。毛细管液相色谱（Capillary LC）可提供更为温和的实验条件，但适配该流速的商用尺寸排阻色谱柱数量极少，且价格高得令众多实验室望而却步。现有的缓冲液交换色谱柱装填方案，要么依赖专用设备，要么装填后的色谱柱仅具备有限的基于尺寸的蛋白质分离能力。本研究开发了可自行装填的微型尺寸排阻色谱柱，其搭载不同固定相且尺寸可定制。本研究采用普通液相色谱泵通过浆料法装填此类色谱柱，并将其应用于多类样品的多项实验中，涵盖天然质谱、自上而下质谱（top-down MS，TDMS）、配体筛选以及离子迁移质谱（ion mobility-MS，IM-MS）等场景。基于天然条件的分离操作，可实现对包含多种蛋白质的样品的数据采集。我们在12分钟内完成了3种蛋白质的自上而下天然质谱数据采集，序列覆盖率最高可达47%。我们在约60分钟内（包含校准品的测试时间）完成了不同电荷状态下α-突触核蛋白的离子迁移质谱检测，所得结果与已有文献报道一致。最后，我们利用SEC-nMS技术快速筛选蛋白水解靶向嵌合体（proteolysis-targeting chimera，PROTAC）候选化合物，并对PROTAC诱导形成的三元复合物开展了碰撞诱导解折叠（collision-induced unfolding，CIU）实验。通过本项研究，我们论证了尺寸排阻色谱在助力结构质谱领域发展方面的巨大潜力。