Abstract
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.
Highlights
Exosomes are usually defined as spherical extracellular nanovesicles from endocytic origin with a diameter between 20 and 150 nm, that are secreted to the extracellular media by almost all cell lines present in the organism
Uncertainties around 10% are usually considered acceptable in general lipidomic analysis using the gold standard electrospray (ESI)-Mass Spectrometry (MS) quantification [3], and uncertainties between 5% and 15% were obtained for most lipid species analyzed in exosomes by different methods combining chromatography and MS [2,4]
Conditions for HPTLC chromatographic development were targeted for separating most PL classes from each other and from other lipids in exosome extracts, and detecting and quantifying by densitometry
Summary
Exosomes are usually defined as spherical extracellular nanovesicles from endocytic origin with a diameter between 20 and 150 nm, that are secreted to the extracellular media by almost all cell lines present in the organism. As several works pointed out, our current knowledge about composition and function of lipids in these vesicles is limited, mostly due to: the weakness of an exosome definition based on size and morphology; the variability of the biological sources from which they are extracted; and the uncertainty of methods for lipid characterization [2] In this regard, uncertainties around 10% are usually considered acceptable in general lipidomic analysis using the gold standard electrospray (ESI)-Mass Spectrometry (MS) quantification [3], and uncertainties between 5% and 15% (or even greater) were obtained for most lipid species analyzed in exosomes by different methods combining chromatography and MS [2,4]. Chromatography (HPTLC), provides efficient separation of sample into lipid classes, detection and quantitative quantification by UV/FL densitometry, using the appropriate standards, and direct coupling with MS. As requested in previous works [1,2], importance has been given here to the description of methodological details used to purify and isolate exosomes, and to analyze their extracted phospholipids, such that the studies can be reproduced
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