Container-less Acoustic Levitation Expands Plasma Extracellular Vesicle Proteome Coverage by Mitigating Size-Dependent Peptide Loss

<jats:p>Proteomic profiling of plasma-derived extracellular vesicles (EVs) is limited in part by adsorptive loss of peptides and proteins to container walls during sample preparation. Here we apply an automated, environment-controlled acoustic levitation platform (Levcell) to the tryptic digestion of small EVs (sEVs) isolated from pooled breast cancer patient plasma, and compare it directly with digestion in low-bind microcentrifuge tubes. Across three parallel technical replicates per method, container-less digestion identified 309 +/- 22 protein groups versus 261 +/- 6 for tubes (+18.4%; Welch t-test p = 0.053), with equivalent or better quantitative reproducibility (median CV 12.0% vs 14.4%). The gain was strongly asymmetric: 66 protein groups were recovered only under levitation while 10 were recovered only in tubes (exact McNemar p = 3 * 10e-11). Peptides recovered exclusively by levitation were longer and heavier than those exclusive to tubes (median 14 vs 12 residues, 1611 vs 1358 Da; p &lt; 2 * 10e-6; Cliff's delta approx. 0.19-0.20), whereas the total peptide pools were indistinguishable and mean missed-cleavage rates were equivalent (0.276 vs 0.263, p = 0.41), excluding differential digestion efficiency as an explanation. No systematic difference in hydropathy, isoelectric point or hydrophobic residue frequency was detected. The levitation-rescued sub-proteome was enriched for ribosomal, proteasomal, chaperonin and RNA-binding complexes - canonical sEV luminal cargo (MYC targets 16/19, odds ratio 31.7, q = 5.6 * 10e-8) - and covered 33 of the 100 ExoCarta reference markers versus 22 for tubes, gaining 14 markers while losing three (a single ezrin/moesin/radixin protein group; McNemar p = 9.8 * 10e-4). We also report two findings that temper the approach: levitated samples carried an approximately 3.7-fold higher keratin burden, consistent with airborne contamination in an open chamber, and no individual marker showed a significant abundance difference after correction for multiple testing. Container-less processing therefore offers a reproducible gain in sEV proteome coverage attributable to reduced size-dependent peptide loss, provided that contamination control is addressed.</jats:p>