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Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcifi...

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Autores principales: Trindade, Fábio, Ferreira, Ana F., Saraiva, Francisca, Martins, Diana, Mendes, Vera M., Sousa, Carla, Gavina, Cristina, Leite-Moreira, Adelino, Manadas, Bruno, Falcão-Pires, Inês, Vitorino, Rui
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505568/
https://www.ncbi.nlm.nih.gov/pubmed/36136308
http://dx.doi.org/10.3390/proteomes10030030
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author Trindade, Fábio
Ferreira, Ana F.
Saraiva, Francisca
Martins, Diana
Mendes, Vera M.
Sousa, Carla
Gavina, Cristina
Leite-Moreira, Adelino
Manadas, Bruno
Falcão-Pires, Inês
Vitorino, Rui
author_facet Trindade, Fábio
Ferreira, Ana F.
Saraiva, Francisca
Martins, Diana
Mendes, Vera M.
Sousa, Carla
Gavina, Cristina
Leite-Moreira, Adelino
Manadas, Bruno
Falcão-Pires, Inês
Vitorino, Rui
author_sort Trindade, Fábio
collection PubMed
description The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex.
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spelling pubmed-95055682022-09-24 Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure Trindade, Fábio Ferreira, Ana F. Saraiva, Francisca Martins, Diana Mendes, Vera M. Sousa, Carla Gavina, Cristina Leite-Moreira, Adelino Manadas, Bruno Falcão-Pires, Inês Vitorino, Rui Proteomes Article The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex. MDPI 2022-09-01 /pmc/articles/PMC9505568/ /pubmed/36136308 http://dx.doi.org/10.3390/proteomes10030030 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Trindade, Fábio
Ferreira, Ana F.
Saraiva, Francisca
Martins, Diana
Mendes, Vera M.
Sousa, Carla
Gavina, Cristina
Leite-Moreira, Adelino
Manadas, Bruno
Falcão-Pires, Inês
Vitorino, Rui
Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure
title Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure
title_full Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure
title_fullStr Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure
title_full_unstemmed Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure
title_short Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure
title_sort optimization of a protocol for protein extraction from calcified aortic valves for proteomics applications: development of a standard operating procedure
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505568/
https://www.ncbi.nlm.nih.gov/pubmed/36136308
http://dx.doi.org/10.3390/proteomes10030030
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