{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T02:19:23Z","timestamp":1788488363740,"version":"build-2803163510"},"reference-count":24,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,1,21]],"date-time":"2022-01-21T00:00:00Z","timestamp":1642723200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u00b5g PL\/100 \u00b5g 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.<\/jats:p>","DOI":"10.3390\/ijms23031150","type":"journal-article","created":{"date-parts":[[2022,1,21]],"date-time":"2022-01-21T08:37:18Z","timestamp":1642754238000},"page":"1150","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells"],"prefix":"10.3390","volume":"23","author":[{"given":"Mar\u00eda","family":"Sancho-Albero","sequence":"first","affiliation":[{"name":"Department of Chemical Engineering, Aragon Institute of Nanoscience (INA), University of Zaragoza, 50018 Zaragoza, Spain"},{"name":"Networking Research Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"Jarne","sequence":"additional","affiliation":[{"name":"Instituto de Carboqu\u00edmica, ICB-CSIC, 50018 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda","family":"Savir\u00f3n","sequence":"additional","affiliation":[{"name":"CEQMA-CSIC, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2890-7846","authenticated-orcid":false,"given":"Pilar","family":"Mart\u00edn-Duque","sequence":"additional","affiliation":[{"name":"Networking Research Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain"},{"name":"Instituto Aragon\u00e9s de Ciencias de la Salud, IIS Arag\u00f3n, 50009 Zaragoza, Spain"},{"name":"Fundaci\u00f3n Araid, 50018, Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis","family":"Membrado","sequence":"additional","affiliation":[{"name":"Instituto de Carboqu\u00edmica, ICB-CSIC, 50018 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9786-9217","authenticated-orcid":false,"given":"Vicente L.","family":"Cebolla","sequence":"additional","affiliation":[{"name":"Instituto de Carboqu\u00edmica, ICB-CSIC, 50018 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jes\u00fas","family":"Santamar\u00eda","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Aragon Institute of Nanoscience (INA), University of Zaragoza, 50018 Zaragoza, Spain"},{"name":"Networking Research Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1016\/j.addr.2020.03.002","article-title":"An emerging focus on lipids in extracellular vesicles","volume":"159","author":"Skotland","year":"2020","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.plipres.2017.03.001","article-title":"Lipids in Exosomes: Current Knowledge and the Way Forward","volume":"66","author":"Skotland","year":"2017","journal-title":"Prog. Lipid Res."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Han, X.L. (2016). Lipidomics. Comprehensive Mass Spectrometry of Lipids, John Wiley & Sons.","DOI":"10.1002\/9781119085263"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1194\/jlr.R084343","article-title":"Exosomal Lipid Composition and the Role of Ether Lipids and Phosphoinositides in Exosome Biology","volume":"60","author":"Skotland","year":"2019","journal-title":"J. Lipid Res."},{"key":"ref_5","first-page":"45","article-title":"Composition, Physicochemical and Biological Properties of Exosomes Secreted From Cancer Cells","volume":"Volume 3","author":"Amiji","year":"2015","journal-title":"Diagnostic and Therapeutic Applications of Exosomes in Cancer"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.ejca.2016.10.011","article-title":"Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers","volume":"70","author":"Skotland","year":"2017","journal-title":"Eur. J. Cancer"},{"key":"ref_7","first-page":"1302","article-title":"Molecular lipidomics of exosomes released by PC-3 prostate cancer cells","volume":"1831","author":"Llorente","year":"2013","journal-title":"Biochim. Biophys. Acta (BBA)\u2014Mol. Cell Biol. Lipids"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"204","DOI":"10.1111\/tra.12803","article-title":"The role of lipids in exosome biology and intercelular communciation: Function, analytics and applications","volume":"22","year":"2021","journal-title":"Traffic"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1016\/j.molmed.2008.08.005","article-title":"Arachidonic-acid-derived eicosanoids: Roles in biology and inmunopathology","volume":"14","author":"Harizi","year":"2008","journal-title":"Trends Mol. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"32570","DOI":"10.3402\/jev.v5.32570","article-title":"High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources","volume":"5","author":"Haraszti","year":"2016","journal-title":"J. Extracell. Vesicles"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Beloribi, S., Ristorcelli, E., Breuzard, G., Silvy, F., Bertrand-Michel, J., Beraud, E., Verine, A., and Lombardo, D. (2012). Exosomal lipids impact notch signaling and induce death of human pancreatic tumoral SOJ-6 cells. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0047480"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1080\/10826076.2020.1866600","article-title":"Scanning densitometry and mass spectrometry for HPTLC analysis of lipids: The last 10 years","volume":"44","author":"Cebolla","year":"2021","journal-title":"J. Liq. Chromatogr. Relat. Technol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"13834","DOI":"10.1038\/s41598-019-50195-z","article-title":"Discrimination of Urinary Exosomes from Microvesicles by Lipidomics Using Thin Layer Liquid Chromatography (TLC) Coupled with MALDI-TOF Mass Spectrometry","volume":"9","author":"Singhto","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_14","first-page":"213","article-title":"TLC\/MALDI MS for the Analysis of Lipids","volume":"Volume 110","author":"Kowalska","year":"2016","journal-title":"Planar Chromatography-Mass Spectrometry"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"461895","DOI":"10.1016\/j.chroma.2021.461895","article-title":"Globotriaosylceramide-related biomarkers of fabry disease identified in plasma by high-performance thin-layer chromatography-densitometry-mass spectrometry","volume":"1638","author":"Jarne","year":"2021","journal-title":"J. Chromatogr. A"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"123001","DOI":"10.1016\/j.jchromb.2021.123001","article-title":"The value of coupling thin-layer chromatography to mass spectrometry in lipid research\u2014A review","volume":"1185","author":"Engel","year":"2021","journal-title":"J. Chromatogr. B"},{"key":"ref_17","first-page":"1157","article-title":"Coupling of planar chromatography to mass spectrometry","volume":"29","author":"Morlock","year":"2010","journal-title":"TrAC"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1177\/1469066718815380","article-title":"Identification of Lipids of the Stratum Corneum by High Performance Thin Layer Chromatography and Mass Spectrometry","volume":"25","author":"Jamin","year":"2019","journal-title":"Eur. J. Mass Spectrom."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/10826076.2018.1561465","article-title":"HPTLC Coupled to ESI-Tandem MS for Identifying Phospholipids Associated to Membrane Proteins in Photosynthetic Purple Bacteria","volume":"42","author":"Lapieza","year":"2019","journal-title":"J. Liq. Chromatogr. Relat. Technol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1002\/cphc.201100590","article-title":"Changes in Fluorescent Emission Due to Non-Covalent Interactions as a General Detection Procedure for Thin-Layer Chromatography","volume":"13","author":"Cebolla","year":"2012","journal-title":"ChemPhysChem"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"167","DOI":"10.3390\/chromatography2020167","article-title":"A Hyphenated Technique Based on High-Performance Thin Layer Chromatography for Determining Neutral Sphingolipids: A Proof of Concept","volume":"2","author":"Jarne","year":"2015","journal-title":"Chromatography"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0021-9258(18)64849-5","article-title":"A simple method for the isolation and purification of total lipids from animal tissues","volume":"226","author":"Folch","year":"1957","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1186\/s12944-018-0854-x","article-title":"Lipidomic profiling of extracellular vesicles derived from prostate and prostate cancer cell lines","volume":"17","author":"Brzozowski","year":"2018","journal-title":"Lipids Health Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.aca.2008.04.043","article-title":"Interferences and Contaminants Encountered in Modern Mass Spectrometry","volume":"627","author":"Keller","year":"2008","journal-title":"Anal. Chim. Acta"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/3\/1150\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:05:04Z","timestamp":1760133904000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/3\/1150"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,21]]},"references-count":24,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["ijms23031150"],"URL":"https:\/\/doi.org\/10.3390\/ijms23031150","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,21]]}}}