Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry

Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol compositi...
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Gespeichert in:

Autor*in:	Ilijana Begcevic Brkovic [verfasserIn] Madlen Reinicke [verfasserIn] Soroth Chey [verfasserIn] Ingo Bechmann [verfasserIn] Uta Ceglarek [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2023

Schlagwörter:	sterols targeted mass spectrometry plasma membrane microdomains microglia plant sterols cholesterol precursors

Übergeordnetes Werk:	In: Cells - MDPI AG, 2012, 12(2023), 7, p 974
Übergeordnetes Werk:	volume:12 ; year:2023 ; number:7, p 974

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.3390/cells12070974

Katalog-ID:	DOAJ089391780

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520			\|a Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.
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allfields	10.3390/cells12070974 doi (DE-627)DOAJ089391780 (DE-599)DOAJ764a9440f01b4b81b16227f8c4be2bd6 DE-627 ger DE-627 rakwb eng QH573-671 Ilijana Begcevic Brkovic verfasserin aut Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling. sterols targeted mass spectrometry plasma membrane microdomains microglia plant sterols cholesterol precursors Cytology Madlen Reinicke verfasserin aut Soroth Chey verfasserin aut Ingo Bechmann verfasserin aut Uta Ceglarek verfasserin aut In Cells MDPI AG, 2012 12(2023), 7, p 974 (DE-627)718622081 (DE-600)2661518-6 20734409 nnns volume:12 year:2023 number:7, p 974 https://doi.org/10.3390/cells12070974 kostenfrei https://doaj.org/article/764a9440f01b4b81b16227f8c4be2bd6 kostenfrei https://www.mdpi.com/2073-4409/12/7/974 kostenfrei https://doaj.org/toc/2073-4409 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2023 7, p 974
spelling	10.3390/cells12070974 doi (DE-627)DOAJ089391780 (DE-599)DOAJ764a9440f01b4b81b16227f8c4be2bd6 DE-627 ger DE-627 rakwb eng QH573-671 Ilijana Begcevic Brkovic verfasserin aut Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling. sterols targeted mass spectrometry plasma membrane microdomains microglia plant sterols cholesterol precursors Cytology Madlen Reinicke verfasserin aut Soroth Chey verfasserin aut Ingo Bechmann verfasserin aut Uta Ceglarek verfasserin aut In Cells MDPI AG, 2012 12(2023), 7, p 974 (DE-627)718622081 (DE-600)2661518-6 20734409 nnns volume:12 year:2023 number:7, p 974 https://doi.org/10.3390/cells12070974 kostenfrei https://doaj.org/article/764a9440f01b4b81b16227f8c4be2bd6 kostenfrei https://www.mdpi.com/2073-4409/12/7/974 kostenfrei https://doaj.org/toc/2073-4409 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2023 7, p 974
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allfieldsSound	10.3390/cells12070974 doi (DE-627)DOAJ089391780 (DE-599)DOAJ764a9440f01b4b81b16227f8c4be2bd6 DE-627 ger DE-627 rakwb eng QH573-671 Ilijana Begcevic Brkovic verfasserin aut Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling. sterols targeted mass spectrometry plasma membrane microdomains microglia plant sterols cholesterol precursors Cytology Madlen Reinicke verfasserin aut Soroth Chey verfasserin aut Ingo Bechmann verfasserin aut Uta Ceglarek verfasserin aut In Cells MDPI AG, 2012 12(2023), 7, p 974 (DE-627)718622081 (DE-600)2661518-6 20734409 nnns volume:12 year:2023 number:7, p 974 https://doi.org/10.3390/cells12070974 kostenfrei https://doaj.org/article/764a9440f01b4b81b16227f8c4be2bd6 kostenfrei https://www.mdpi.com/2073-4409/12/7/974 kostenfrei https://doaj.org/toc/2073-4409 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2023 7, p 974
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callnumber-first	Q - Science
author	Ilijana Begcevic Brkovic
spellingShingle	Ilijana Begcevic Brkovic misc QH573-671 misc sterols misc targeted mass spectrometry misc plasma membrane microdomains misc microglia misc plant sterols misc cholesterol precursors misc Cytology Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry
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topic_title	QH573-671 Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry sterols targeted mass spectrometry plasma membrane microdomains microglia plant sterols cholesterol precursors
topic	misc QH573-671 misc sterols misc targeted mass spectrometry misc plasma membrane microdomains misc microglia misc plant sterols misc cholesterol precursors misc Cytology
topic_unstemmed	misc QH573-671 misc sterols misc targeted mass spectrometry misc plasma membrane microdomains misc microglia misc plant sterols misc cholesterol precursors misc Cytology
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title	Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry
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title_full	Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry
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title_sort	characterization of non-cholesterol sterols in microglia cell membranes using targeted mass spectrometry
callnumber	QH573-671
title_auth	Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry
abstract	Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.
abstractGer	Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.
abstract_unstemmed	Background: Non-cholesterol sterols, as well as plant sterols, cross the blood–brain barrier and, thus, can be incorporated into cell membranes, affecting the cell’s inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep<sup<TM</sup< gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4–6, <i<n</i< = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.
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title_short	Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry
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The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE<LA<SI<ST<CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. 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Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry

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