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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| Format: | Article |
| Language: | English |
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MDPI AG
2021-10-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/20/11144 |
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| author | Mikel Azkargorta Ibon Iloro Iraide Escobes Diana Cabrera Juan M. Falcon-Perez Felix Elortza Felix Royo |
| author_facet | Mikel Azkargorta Ibon Iloro Iraide Escobes Diana Cabrera Juan M. Falcon-Perez Felix Elortza Felix Royo |
| author_sort | Mikel Azkargorta |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T06:30:01Z |
| format | Article |
| id | doaj.art-ad4af7b396844502ba0be321555327a0 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T06:30:01Z |
| publishDate | 2021-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-ad4af7b396844502ba0be321555327a02023-11-22T18:34:39ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-10-0122201114410.3390/ijms222011144Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method EmployedMikel Azkargorta0Ibon Iloro1Iraide Escobes2Diana Cabrera3Juan M. Falcon-Perez4Felix Elortza5Felix Royo6Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160 Derio, SpainThe 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.https://www.mdpi.com/1422-0067/22/20/11144extracellular vesiclesultracentrifugationsize exclusionprecipitation kitsproteomicserum |
| spellingShingle | Mikel Azkargorta Ibon Iloro Iraide Escobes Diana Cabrera Juan M. Falcon-Perez Felix Elortza Felix Royo Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed International Journal of Molecular Sciences extracellular vesicles ultracentrifugation size exclusion precipitation kits proteomic serum |
| 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 | extracellular vesicles ultracentrifugation size exclusion precipitation kits proteomic serum |
| url | https://www.mdpi.com/1422-0067/22/20/11144 |
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