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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...

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Autores principales: Sancho-Albero, María, Jarne, Carmen, Savirón, María, Martín-Duque, Pilar, Membrado, Luis, Cebolla, Vicente L., Santamaría, Jesús
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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