Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers
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
Affiliations
Laura Martelo-Vidal
BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Sara Vázquez-Mera
BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Pablo Miguéns-Suárez
BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Susana Belén Bravo-López
Proteomic Service, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain
Heidi Makrinioti
Department of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
Vicente Domínguez-Arca
Grupo de Física de Coloides y Polímeros, Departamento de Física de Partículas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Javier de-Miguel-Díez
Respiratory Department, Hospital General Universitario Gregorio Marañón, 28009 Madrid, Spain
Alberto Gómez-Carballa
Genetics, Vaccines and Infections Research Group (GenViP), Instituto de Investigación Sanitaria de Santiago, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain
Antonio Salas
Genetics, Vaccines and Infections Research Group (GenViP), Instituto de Investigación Sanitaria de Santiago, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain
Francisco Javier González-Barcala
BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Francisco Javier Salgado
BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Juan José Nieto-Fontarigo
BioLympho Research Group, Department of Biochemistry and Molecular Biology, Faculty of Biology-Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
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 high 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.
