Quantitative Proteomic Analysis Unveils Protein Concentration Effects in Neat Urine Compared to Urine Extracellular Vesicles

Urine proteomics and extracellular vesicle (EV) research have emerged as promising fields for biomarker discovery. While urine protein concentration is known to influence proteomics and EV isolation, its effect on both of these combined remains unclear. This study compares proteomic signatures between neat urine and EV-enriched urine in two protein concentration groups (≤0.50 and >0.5 g/L) using two EV enrichment methods. Data-independent acquisition (DIA) was employed for mass spectrometry (MS) proteomic analysis. At low-protein concentrations, neat urine yielded a richer proteome, identifying more proteins, including EV-associated proteins, compared to enriched urine. At high-protein concentrations, neat urine showed reduced protein identification, while enrichment improved the detection of unique proteins and decreased the relative abundance of high-abundance proteins. These findings suggest that, based on (MS)-based proteomics with DIA (DIA-MS), neat urine is a superior source of potential biomarkers at low-protein concentrations, whereas at high-protein concentrations, EV enrichment effectively standardizes signatures for better proteome detection. This study highlights the impact of urine protein concentration on discovery proteomics and the importance of tailoring methodologies to different sample characteristics in biomarker discovery.