Electrochemical profiling of urinary extracellular vesicle proteomes to reveal differential hypoxic adaptation

Hypobaric hypoxia induces physiological stress, yet early molecular alterations preceding functional impairment are not adequately captured by conventional physiologic analysis. Extracellular vesicles (EVs), encapsulated proteins and RNAs reflective of cellular state, represent a compelling source of non-invasive biomarkers for interrogating hypoxia-induced molecular adaptation at the proteome and post-transcriptional levels. Here, we present an electrochemical-proteomic framework for quantitative profiling of urinary EV (uEV) cargo during hypobaric hypoxic exposure. Two analog astronauts were monitored during a 10-day high-altitude expedition (14,860 ft). Pre-, mid-, and post-exposure physiological assessments using pulse oximetry and spirometry were complemented by molecular analysis of uEVs. EVs were captured directly on a screen-printed gold electrode-based immunosensor, followed by electric-field lysis for offering on-chip access to intravesicular cargo. EV enrichment and hypoxic stress responses were validated by Western blotting. Non-faradaic electrochemical impedance spectroscopy enables quantification of EV-associated proteins, including epithelial secretory proteins (MUC5AC and MUC1), EV biogenesis markers (TSG101), membrane remodeling proteins (GM2AP), and growth factor signaling receptors (HER2, EGFR, and GPC1), while EV-associated microRNAs were profiled by small RNA sequencing. The participant exhibiting greater physiological perturbation under hypoxia showed reduced pulmonary function and coordinated upregulation of EV proteins linked to epithelial activation, vesicle biogenesis, membrane remodeling, and growth-factor signaling, along with microRNAs (miR-200 family, miR-375, and the miR-17-92 cluster). In contrast, the participant maintaining more stable pulmonary function under hypoxia exhibited stable lung mechanics with minimal molecular perturbation. These findings underscore electrochemical uEV profiling as a sensitive and non-invasive approach for proteome-level assessment of individual-specific physiological response to hypoxic stress under extreme environmental conditions.