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 remains 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 were stably levitated and allowed to evaporate midair, without active acceleration, using an asymmetric acoustic levitator to concentrate analytes prior to CE-MS analysis. Optical imaging confirmed a ∼4.4-fold volume reduction after 60 min of passive evaporation, corresponding to ∼3.4-fold concentration enrichment based on label-free quantification of the resulting proteome. This enhancement increased sample utilization by ∼100% compared to conventional CE-ESI-MS, doubling protein identifications from ∼3 ng HeLa digest (∼10 cells) to 2169 proteins after enrichment (∼14 ng). Quantitation showed consistent proteome composition without concentration or hydrophobicity bias. These results establish Levitational CE-ESI-MS as a sensitive, reproducible, and contact-free strategy for trace-level proteomics, opening new possibilities for low-input including single-cell analyses.