RT Journal Article
T1 Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers
A1 Martelo Vidal, Laura
A1 Vázquez Mera, Sara
A1 Miguéns Suárez, Pablo
A1 Domínguez Arca, Vicente
A1 Gómez Carballa, Alberto
A1 Salas Ellacuriaga, Antonio
A1 González Barcala, Francisco Javier
A1 Salgado Castro, Francisco Javier
A1 Nieto Fontarigo, Juan José
K1 Exosomes
K1 MiRNAs
K1 Proteomics
K1 Respiratory disease biomarkers
K1 Urine
AB This study aims to develop a protocol for respiratory disease-associated biomarker discovery by combining urine proteome studies with urinary exosome components analysis (i.e., miRNAs). To achieve this, urine was DTT treated to decrease uromodulin, then concentrated and ultracentrifuged. Proteomic analyses of exosome-free urine were performed using LC-MS/MS. Simultaneously, miRNA expression from urine exosomes was measured using either RTqPCR (pre-amplification) or nCounter Nanostring (non-amplication) analyses. We detected 548 different proteins in exosome-free urine samples (N = 5) with high confidence (FDR < 1%), many of them being expressed in different non-renal tissues. Specifically, lung-related proteins were overrepresented (Fold enrichment = 1.31; FDR = 0.0335) compared to whole human proteome, and 10–15% were already described as protein biomarkers for several pulmonary diseases. Urine proteins identified belong to several functional categories important in respiratory pathology. We could confirm the expression of miRNAs previously connected to respiratory diseases (i.e., miR-16-5p, miR-21-5p, miR-146a-5p, and miR-215-5p) in urine exosomes by RTqPCR. Finally, we detected 333 miRNAs using Nanostring, 15 of them up-regulated in T2high asthma (N = 4) compared to T2low asthma (N = 4) and healthy subjects (N = 4). Therefore, this protocol combining the urinary proteome (exosome free) with the study of urinary exosome components (i.e., miRNAs) holds great potential for molecular biomarker discovery of non-renal and particularly respiratory pathologies.
PB Multidisciplinary Digital Publishing Institute (MDPI)
SN 2218-273X
YR 2025
FD 2025-01-01
LK https://hdl.handle.net/10347/46317
UL https://hdl.handle.net/10347/46317
LA eng
NO Martelo-Vidal, L., Vázquez-Mera, S., Miguéns-Suárez, P., Bravo-López, S. B., Makrinioti, H., Domínguez-Arca, V., de-Miguel-Díez, J., Gómez-Carballa, A., Salas, A., González-Barcala, F. J., Salgado, F. J., & Nieto-Fontarigo, J. J. (2025). Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers. Biomolecules, 15(1). https://doi.org/10.3390/BIOM15010060
NO This work has been supported by grants from Sociedad Española de Neumología y Cirugía Torácica (SEPAR, 938/2019, PROTRANSUR), and Instituto de Salud Carlos III (ISCIII)/PI22/00502/cofinanced by the European Union. The publication is part of the grant RYC2021-032676-I financed by MCIN/AEI/10.13039/501100011033 and by the European Union NextGeneration EU/PRTR. SV-M and PM-S are recipients of a Xunta de Galicia PhD Fellowship (co-financed by European Social Fund, ESF).
DS Minerva
RD 23 may 2026