Online Coupling of Acoustic Droplet Levitation with Capillary Electrophoresis Mass Spectrometry Enables Midair Enrichment for Improved Proteomic Sensitivity

Contact between analytes and surfaces during sample handling is a major barrier to sensitivity in trace-level proteomics, including single-cell mass spectrometry (MS). Here, we introduce the first online integration of acoustic droplet levitation with capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS), enabling containerless, midair sample enrichment and analysis. In this levitational CE-MS platform, droplets containing proteome digests are stably floated and can be evaporated midair using an asymmetric acoustic levitator to concentrate analytes before CE-MS analysis. Optical imaging confirmed a ~4.4-fold volume reduction after 60 min of evaporation, translating to ~3.4-fold concentration enrichment based on label-free quantification of the detected proteins. This enrichment enhanced sample utilization by ~100% compared to conventional CE-ESI-MS. The number of identified proteins were nearly doubled from ~3 ng of HeLa digest, approximating to ~10 cells, with 2,169 proteins detected after enrichment midair compared to the levitated-only control. Quantitative analysis revealed consistent composition and no detectable bias in concentration or hydrophobicity for the measured peptides. Our results establish levitational CE-ESI-MS as a sensitive, reproducible, and containerless strategy for trace proteomics, opening new avenues for low-input, including single-cell, analyses.