Fast tracking native mass spectrometry: Skipping over buffer exchange

Post-translational modifications play an important role in eukaryotic protein structures and their interactions, which often cannot be achieved in bacterial expression systems such as E . coli . However, it often proves to be quite challenging to produce large amounts of pure protein for downstream analysis like native mass spectrometry (nMS). This is due to lower expression rates in eukaryotic expression systems, such as insect or mammalian cells, compared to bacterial expression systems. Moreover, nMS protocols typically include a buffer exchange step that often results in substantial protein loss, especially when the input material is limited. This buffer exchange step is needed because standard lysis and purification buffers contain Tris, phosphate, or HEPES buffer supplemented with sodium chloride. These are not suitable for nMS as they are non-volatile salts that form adducts during electrospray ionization (ESI), resulting in interference with the signal. Here, we developed a novel method that eliminates the buffer exchange step after protein purification to allow nMS measurements by directly eluting the proteins of interest with an ammonium-acetate-based buffer. We show that the presence of common eluents used in affinity purification protocols, such as imidazole, biotin, and desthiobiotin, is compatible with obtaining high-quality spectra to derive protein stoichiometry and even analyzing enzymatic processes.