Year: 2025
Type: article
Abstract: Slow mixing mode native mass spectrometry (SLOMO-nMS), which monitors the mixing of layered solutions within a nanoflow electrospray ionization (nanoESI) emitter, enables the accurate quantification of biomolecular complexes in vitro, even when absolute concentrations are unknown. The method relies on mass balance principles, assuming that the total concentration of one of the 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 nonuniform 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 electrophoretic motion. Our results reveal that diffusion is the primary driver of mixing and, with advection, bulk transport. In contrast, electrophoretic displacement of the analyte is negligible at typical nanoESI voltages. Notably, the effective flow rate associated with analyte diffusion, quantified here for the first time, is found to be comparable to the overall solution flow rates under low-voltage conditions; at higher voltages, advection dominates analyte transport in the emitter. Together, these findings provide support for the mass balance assumptions underlying SLOMO-nMS and have broader implications for other long-duration nMS experiments that rely on a stable solution-phase composition.
Authors: Simar K. Dhillon, Duong T. Bui, Elena N. Kitova, Lara K. Mahal, John S. Klassen
Institution: University of Alberta
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W4415313923