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High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells
The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8835402/ https://www.ncbi.nlm.nih.gov/pubmed/35163074 http://dx.doi.org/10.3390/ijms23031150 |
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author | Sancho-Albero, María Jarne, Carmen Savirón, María Martín-Duque, Pilar Membrado, Luis Cebolla, Vicente L. Santamaría, Jesús |
author_facet | Sancho-Albero, María Jarne, Carmen Savirón, María Martín-Duque, Pilar Membrado, Luis Cebolla, Vicente L. Santamaría, Jesús |
author_sort | Sancho-Albero, María |
collection | PubMed |
description | The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface. |
format | Online Article Text |
id | pubmed-8835402 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-88354022022-02-12 High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells Sancho-Albero, María Jarne, Carmen Savirón, María Martín-Duque, Pilar Membrado, Luis Cebolla, Vicente L. Santamaría, Jesús Int J Mol Sci Article The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface. MDPI 2022-01-21 /pmc/articles/PMC8835402/ /pubmed/35163074 http://dx.doi.org/10.3390/ijms23031150 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 Sancho-Albero, María Jarne, Carmen Savirón, María Martín-Duque, Pilar Membrado, Luis Cebolla, Vicente L. Santamaría, Jesús High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells |
title | High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells |
title_full | High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells |
title_fullStr | High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells |
title_full_unstemmed | High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells |
title_short | High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells |
title_sort | high-performance thin-layer chromatography-densitometry-tandem esi-ms to evaluate phospholipid content in exosomes of cancer cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8835402/ https://www.ncbi.nlm.nih.gov/pubmed/35163074 http://dx.doi.org/10.3390/ijms23031150 |
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