Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH com-bined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-mu L/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-

Automated summary

Size-exclusion chromatography (SEC) with volatile salts and native mass spectrometry (nMS) is valuable for characterizing proteins and aggregates. However, high salt concentrations in SEC-nMS hinder the analysis of labile protein complexes in the gas phase. To overcome this, narrow SEC columns operated at reduced flow rates were used, resulting in improved protein ionization efficiency and the detection of low-abundant impurities and higher-order structures. Lower gas temperatures ensured minimal structural alterations during transfer into the gas phase, and decreased ionization suppression allowed the use of volatile solvents.