Simple urine storage protocol for extracellular vesicle proteomics compatible with at-home self-sampling

Area of Science:

• Biochemistry
• Biomarker Discovery
• Urology

Background:

• Urinary extracellular vesicles (EVs) are promising biomarkers.
• Clinical use requires reliable short-term storage protocols, especially for at-home collections.
• The impact of delayed processing on urinary EV concentration and proteome is not well understood.

Purpose of the Study:

• To evaluate the stability of urinary EVs under different short-term storage conditions.
• To assess the effect of storage duration and EDTA preservative on EV concentration and proteome.
• To determine the suitability of urine storage for at-home collection and subsequent biomarker analysis.

Main Methods:

• Urine samples were stored at 4°C and at room temperature with EDTA.
• Storage durations included 0, 2, 4, and 8 days.
• EVs were isolated using the ME-kit (VN96-peptide).
• EV concentration was measured using EVQuant.
• Proteome analysis was performed using data-independent acquisition mass spectrometry.

Main Results:

• Urinary EV concentration and proteome remained highly stable under both storage conditions.
• No significant changes in protein number or quantitative proteomic profiles were observed.
• EDTA did not affect urinary EV concentration or the global proteome.
• Storage for up to 8 days showed no significant decay in EV concentration or notable effects on the EV-proteome.

Conclusions:

• Urine can be stored at 4°C or with EDTA at room temperature for up to 8 days for EV analysis.
• These storage protocols support the use of self-collected urine samples for biomarker studies.
• Findings facilitate the clinical implementation of urinary EV biomarkers, particularly in home-based settings.