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.