Reduced Pressure Ionization Enhances Native Mass Spectrometry of Proteins and Protein Complexes

Abstract Native mass spectrometry (MS) enables the analysis of protein interactions in complex biological mixtures. However, nonvolatile salts and buffers commonly present in such samples can cause ion adduction, peak broadening, and reduced signal intensity. Reducing the pressure surrounding the ionization emitter significantly improves native MS performance under these challenging conditions. Signal enhancements of up to 20‐fold were observed with nanoscale emitters, and up to 7‐fold with microscale emitters in high‐salt solutions. Protein ions remained detectable in solutions containing up to 300 mM NaCl, unlike ambient pressure ionization. High signal‐to‐noise was observed for the DDB1:DCAF1 complex at 50 nM using reduced pressure ionization, whereas no readily assignable signal was detected at ambient pressure, demonstrating its utility for detecting tightly bound complexes at trace levels. Coupling to native ion mobility mass spectrometry showed that arrival time distributions and collision cross sections did not depend significantly on the pressure used, indicating that structural information is preserved. These results show that reduced pressure ionization improves native MS performance under conditions that typically suppress signal at ambient pressure, such as high salt or low analyte concentration. The method is compatible with both nano‐ and microscale emitters and requires only minor modifications to existing instrumentation. Reduced pressure ionization expands the range of conditions accessible by native MS and is expected to enable automated, high‐throughput workflows in structural proteomics, biopharmaceutical characterisation, and protein‐ligand interaction studies.