Micro-flow Size-Exclusion Chromatography for enhanced native Mass Spectrometry of proteins and protein complexes

Native Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a useful tool for the characterization of proteins in their native state. However, in many cases the conditions needed to realize the hyphenation of SEC with MS require relative high activation energy and therefore hinder the analysis of labile protein complexes. In this work, we are investigating the advantages of narrow SEC columns (1 mm internal diameter) operated at 15 μL/min flow rates coupled directly to nMS for the characterization of proteins, labile protein complexes and their higher-order structures (HOS). Reducing the flow rate, allowed for a significant increase of the MS sensitivity and ionization efficiency, facilitating detection of low-abundant impurities and HOS (up to the limit of the Orbitrap-MS used, i.e. 230 kDa). More-efficient solvent evaporation could be achieved, allowing using softer MS conditions (e.g. lower gas temperature, lower activation energy) that ensured (little or) no structural alterations or denaturation of the proteins and their HOS during their transfer to the gas phase. Furthermore, high-ionic-strength conditions of volatile salts (200-400 mM), are often necessary to ensure (almost) interaction-free SEC analysis of proteins, such as antibodies (mAbs). With this approach the salt tolerance of the MS was much improved. Because of the reduced column dimensions, band broadening effects resulting from the injection volume became more critical. At high injection volumes (exceeding 3% of the column volume) of more dilute samples, the peak shape and width was affected. Therefore, a new set-up was developed to pre-concentrate the injected proteins on an anion and cation-exchange mixed bed trap column prior to SEC-nMS analysis. This “trap-and-elute” set-up was able to eliminate adverse injection-volume effects in SEC and provide additional desalting, while improving MS detection limits.