Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers
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 a...
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2025-01-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/15/1/60 |
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| author | Laura Martelo-Vidal Sara Vázquez-Mera Pablo Miguéns-Suárez Susana Belén Bravo-López Heidi Makrinioti Vicente Domínguez-Arca Javier de-Miguel-Díez Alberto Gómez-Carballa Antonio Salas Francisco Javier González-Barcala Francisco Javier Salgado Juan José Nieto-Fontarigo |
| author_facet | Laura Martelo-Vidal Sara Vázquez-Mera Pablo Miguéns-Suárez Susana Belén Bravo-López Heidi Makrinioti Vicente Domínguez-Arca Javier de-Miguel-Díez Alberto Gómez-Carballa Antonio Salas Francisco Javier González-Barcala Francisco Javier Salgado Juan José Nieto-Fontarigo |
| author_sort | Laura Martelo-Vidal |
| collection | DOAJ |
| description | 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 T2<sup>high</sup> asthma (N = 4) compared to T2<sup>low</sup> 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. |
| format | Article |
| id | doaj-art-cacb3f1e5359432789fd472d1bf06963 |
| institution | Kabale University |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
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| series | Biomolecules |
| spelling | doaj-art-cacb3f1e5359432789fd472d1bf069632025-01-24T13:25:01ZengMDPI AGBiomolecules2218-273X2025-01-011516010.3390/biom15010060Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases BiomarkersLaura Martelo-Vidal0Sara Vázquez-Mera1Pablo Miguéns-Suárez2Susana Belén Bravo-López3Heidi Makrinioti4Vicente Domínguez-Arca5Javier de-Miguel-Díez6Alberto Gómez-Carballa7Antonio Salas8Francisco Javier González-Barcala9Francisco Javier Salgado10Juan José Nieto-Fontarigo11BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainBioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainBioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainProteomic Service, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, SpainDepartment of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USAGrupo de Física de Coloides y Polímeros, Departamento de Física de Partículas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainRespiratory Department, Hospital General Universitario Gregorio Marañón, 28009 Madrid, SpainGenetics, Vaccines and Infections Research Group (GenViP), Instituto de Investigación Sanitaria de Santiago, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, SpainGenetics, Vaccines and Infections Research Group (GenViP), Instituto de Investigación Sanitaria de Santiago, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, SpainBioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainBioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainBioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SpainThis 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 T2<sup>high</sup> asthma (N = 4) compared to T2<sup>low</sup> 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.https://www.mdpi.com/2218-273X/15/1/60exosomesmiRNAsproteomicsrespiratory disease biomarkersurine |
| spellingShingle | Laura Martelo-Vidal Sara Vázquez-Mera Pablo Miguéns-Suárez Susana Belén Bravo-López Heidi Makrinioti Vicente Domínguez-Arca Javier de-Miguel-Díez Alberto Gómez-Carballa Antonio Salas Francisco Javier González-Barcala Francisco Javier Salgado Juan José Nieto-Fontarigo Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers Biomolecules exosomes miRNAs proteomics respiratory disease biomarkers urine |
| title | Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers |
| title_full | Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers |
| title_fullStr | Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers |
| title_full_unstemmed | Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers |
| title_short | Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers |
| title_sort | urinary proteome and exosome analysis protocol for the discovery of respiratory diseases biomarkers |
| topic | exosomes miRNAs proteomics respiratory disease biomarkers urine |
| url | https://www.mdpi.com/2218-273X/15/1/60 |
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