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Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific s...

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Autores principales: Azkargorta, Mikel, Iloro, Ibon, Escobes, Iraide, Cabrera, Diana, Falcon-Perez, Juan M., Elortza, Felix, Royo, Felix
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8540106/
https://www.ncbi.nlm.nih.gov/pubmed/34681804
http://dx.doi.org/10.3390/ijms222011144
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author Azkargorta, Mikel
Iloro, Ibon
Escobes, Iraide
Cabrera, Diana
Falcon-Perez, Juan M.
Elortza, Felix
Royo, Felix
author_facet Azkargorta, Mikel
Iloro, Ibon
Escobes, Iraide
Cabrera, Diana
Falcon-Perez, Juan M.
Elortza, Felix
Royo, Felix
author_sort Azkargorta, Mikel
collection PubMed
description The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.
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spelling pubmed-85401062021-10-24 Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed Azkargorta, Mikel Iloro, Ibon Escobes, Iraide Cabrera, Diana Falcon-Perez, Juan M. Elortza, Felix Royo, Felix Int J Mol Sci Article The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex. MDPI 2021-10-15 /pmc/articles/PMC8540106/ /pubmed/34681804 http://dx.doi.org/10.3390/ijms222011144 Text en © 2021 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
Azkargorta, Mikel
Iloro, Ibon
Escobes, Iraide
Cabrera, Diana
Falcon-Perez, Juan M.
Elortza, Felix
Royo, Felix
Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
title Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
title_full Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
title_fullStr Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
title_full_unstemmed Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
title_short Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
title_sort human serum extracellular vesicle proteomic profile depends on the enrichment method employed
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8540106/
https://www.ncbi.nlm.nih.gov/pubmed/34681804
http://dx.doi.org/10.3390/ijms222011144
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