Self-packed size-exclusion columns enable versatile high-throughput native, top-down, and ion mobility-mass...

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 incompatible with 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 min, with up to 47 % sequence coverage. IM-MS of alpha-synuclein at different charge states was measured in ca. 60 min (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.