Going with the Flow: Mechanistic Insights into Slow Mixing Mode Native Mass Spectrometry

Slow mixing mode native mass spectrometry (SLOMO-nMS), which monitors the mixing of layered solutions within a nanoflow electrospray ionization (nanoESI) emitter, enables robust quantification of biomolecular complexes in vitro, even when absolute concentrations are unknown. The method relies on mass balance principles, assuming that the concentration of one of interacting species remains constant throughout the mixing process. While this condition is typically achieved by using identical starting concentrations in both solutions, deviations may arise due to non-uniform mass transport within the emitter. Here, we report the first quantitative investigation of the factors governing solution mixing and analyte transport in a nanoESI emitter under an applied electric field. Using a dual-emitter setup and a panel of dyes varying in size and charge, we dissected the contributions of diffusion, advection, and electrophoresis. Our results reveal that diffusion is the primary driver of mixing and, with advection, bulk transport. In contrast, electrophoretic displacement of analyte is negligible at typical nanoESI voltages. Notably, the effective flow rate associated with diffusion is comparable to the overall solution flow rates under low-voltage conditions, highlighting the importance of low-flow regimes for maintaining steady-state concentrations. These findings provide mechanistic validation for the mass balance assumptions underlying SLOMO-nMS and have broader implications for other long-duration nMS experiments that rely on stable solution-phase composition.