Shotgun lipidomics affords comprehensive and quantitative analysis of lipid species in cells and tissues at high-throughput [1 5]. The methodology is based on direct infusion of lipid extracts by electrospray ionization (ESI) combined with tandem mass spectrometry (MS/MS) and/or high resolution Fourier transform mass spectrometry (FTMS) for identification and quantification of lipid species [6]. Shotgun lipidomics affords extensive lipidome coverage by combining the analysis of lipid extracts in positive and negative ion mode [1, 3]. Notably, sterols such as cholesterol and ergosterol exhibit low ionization efficiency in ESI [7]. For this reason, chemical derivatization procedures including acetylation [8] or sulfation [9] are commonly implemented to facilitate ionization, detection and quantification of sterols for global lipidome analysis [1-3, 10]

Casanovas, Albert

Ejsing, Christer S

Hannibal-Bach, Hans Kristian

Jensen, Ole Nørregaard

PubMed

Albert Casanovas

Hans Kristian Hannibal‐Bach

Ole N. Jensen

Christer S. Ejsing

Crossref

European Journal of Lipid Science and Technology

Shotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivity: Recommendation for large‐scale global lipidome analysis

Syddansk Universitets Forskerportal

       University of Southern DenmarkShotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivityRecommendation for large-scale global lipidome analysisCasanovas, Albert; Hannibal-Bach, Hans Kristian; Jensen, Ole Nørregaard; Ejsing, Christer SPublished in:European Journal of Lipid Science and TechnologyDOI:10.1002/ejlt.201400451Publication date:2014Document version:Final published versionCitation for pulished version (APA):Casanovas, A., Hannibal-Bach, H. K., Jensen, O. N., & Ejsing, C. S. (2014). Shotgun lipidomic analysis ofchemically sulfated sterols compromises analytical sensitivity: Recommendation for large-scale global lipidomeanalysis. European Journal of Lipid Science and Technology, 116(12), 1618-1620.https://doi.org/10.1002/ejlt.201400451Go to publication entry in University of Southern Denmark's Research PortalTerms of useThis work is brought to you by the University of Southern Denmark.Unless otherwise specified it has been shared according to the terms for self-archiving.If no other license is stated, these terms apply:            • You may download this work for personal use only.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying this open access versionIf you believe that this document breaches copyright please contact us providing details and we will investigate your claim.Please direct all enquiries to puresupport@bib.sdu.dkDownload date: 20. Oct. 2025Letter to the EditorShotgun lipidomic analysis of chemically sulfated sterolscompromises analytical sensitivity: Recommendation forlarge‐scale global lipidome analysisAlbert Casanovas, Hans Kristian Hannibal‐Bach, Ole N. Jensen and Christer S. EjsingDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, DenmarkKeywords: Sensitivity / Shotgun lipidomics / Sterol sulfationReceived: September 12, 2014 / Accepted: September 26, 2014DOI: 10.1002/ejlt.201400451Shotgun lipidomics affords comprehensive and quantitativeanalysis of lipid species in cells and tissues at high‐throughput [1–5]. The methodology is based on directinfusion of lipid extracts by electrospray ionization (ESI)combined with tandem mass spectrometry (MS/MS) and/orhigh resolution Fourier transform mass spectrometry(FTMS) for identification and quantification of lipidspecies [6]. Shotgun lipidomics affords extensive lipidomecoverage by combining the analysis of lipid extracts in positiveand negative ion mode [1, 3]. Notably, sterols such ascholesterol and ergosterol exhibit low ionization efficiency inESI [7]. For this reason, chemical derivatization proceduresincluding acetylation [8] or sulfation [9] are commonlyimplemented to facilitate ionization, detection and quantifi-cation of sterols for global lipidome analysis [1–3, 10].In the course of large‐scale lipidomic analyses using anLTQ Orbitrap XL mass spectrometer (Thermo Scientific)equipped with a robotic nanoESI source TriVersa NanoMate(Advion Biosciences), we observed a pronounced decreasein the sensitivity of negative ion mode analysis followingrepeated injections of samples subjected to chemical sulfation.This decrease in sensitivity was observed only for negative ionmode analysis of lipid species in underivatized lipid extracts.No decrease in sensitivity was observed for negative ion modeanalysis of chemically sulfated sterols or for positive ion modeanalysis of lipid species in underivatized lipid extracts.To investigate the decrease in analytical sensitivity in furtherdetail we (i) spiked a yeast lysate with internal standards andexecuted two‐step lipid extraction as previously described [3]for the partition of apolar and polar lipids, (ii) subjected theapolar lipid extract (containing sterols) to chemical sulfation[2, 9], and (iii) infused the sulfated sample in batches of tenrepeated injections and monitored the intensity of chemicallysulfated sterols by negative ion mode FTMS. To monitor thedecrease in sensitivity we analyzed the underivatized polar lipidextract [containing phosphatidylinositol (PI) as well as otherpolar lipids [3]] by negative ion mode FTMS before and aftereach batch of ten repeated injections of the sulfated sample.This analysis showed a progressive loss of sensitivity fornegative ion mode analysis of lipid species in the under-ivatized polar lipid extract. To exemplify this effect, theintensity of PI 34:1, an abundant glycerophospholipid speciesin yeast [3, 10], was reduced more than 20‐fold after 30injections of the sulfated sample (Fig. 1a). More importantly,since an intensity threshold is routinely used in shotgunlipidomics data processing [11], the decrease in sensitivitytranslated into a decrease in the number of lipid speciesidentified and quantified (data not shown). Notably, theintensity of chemically sulfated sterols was unaffected(Fig. 1b). As we had previously observed that the sensitivityof positive ion mode analysis was not affected, we transientlyswitched the polarity to positive for 1 h and subsequentlyreverted it to evaluate whether the sensitivity of negative ionmode analysis was restored. This polarity switch partiallyrestored the sensitivity (>40%, in the case of PI 34:1) fornegative ion mode analysis of lipid species in the under-ivatized polar lipid extract (Fig. 1a). Based on these results,we recommend that global shotgun lipidomic experimentsemploying chemical sulfation of sterols use a specificsequence of MS analyses where the analysis of sulfatedsamples is scheduled at the end of all other analyses in ordernot to compromise analytical sensitivity (Fig. 2).In conclusion, here we: (i) demonstrate that shotgunlipidomic analysis of sample extracts subjected to chemicalsulfation can prompt a pronounced loss of sensitivity fornegative ion mode analysis of underivatized lipid extracts;Correspondence: Christer S. Ejsing, Department of Biochemistry andMolecular Biology, University of Southern Denmark, Campusvej 55, 5230Odense, DenmarkE‐mail: cse@bmb.sdu.dkFax: þ45 6550 2467Abbreviations: ESI, electrospray ionization;FTMS,Fourier transformmassspectrometry; MS/MS, tandem mass spectrometry; PI, phosphatidylinositol1618 Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.www.ejlst.comThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, whichpermits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercialand no modifications or adaptations are made.(ii) show that changing polarity is a way to overcome thisanalytical problem; and (iii) recommend that analysis ofchemically sulfated lipid extracts is scheduled at the end ofall other analyses in order not to compromise sensitivity anddata quality of large‐scale global lipidome analyses.We are grateful to Maria Carvalho for providing the protocolfor chemical sulfation of sterol lipids. This work was supported byLundbeckfonden (R44‐A4342, R54‐A5858, CSE), the DanishCouncil for Independent Research |Natural Sciences (10‐094213,AC; 09‐072484, CSE), and a Marie Curie Intra EuropeanFellowship (AC).The authors have declared no conflict of interest.References[1] Sampaio, J. L., Gerl, M. J., Klose, C., Ejsing, C. S., et al.,Membrane lipidome of an epithelial cell line. Proc. Natl.Acad. Sci. USA 2011, 108, 1903–1907.[2] Carvalho, M., Sampaio, J. L., Palm, W., Brankatschk, M.,et al., Effects of diet and development on the Drosophilalipidome. Mol. Syst. Biol. 2012, 8, 600.[3] Ejsing, C. S., Sampaio, J. L., Surendranath, V., Duchoslav,E., et al., Global analysis of the yeast lipidome by quantitativeshotgun mass spectrometry. Proc. Natl. Acad. Sci. USA 2009,106, 2136–2141.Figure 1. (a) Sensitivity of negative ion mode analysis of lipid species in the underivatized polar lipid extract is reduced after analysisof chemically sulfated sterols. The underivatized polar lipid extract was infused using 0.005% (w/v) methylamine in chloroform/methanol(1:5, v/v), and analyzed for 5min by negative ion mode FTMS analysis. The intensity of PI 34:1 is shown as a representative lipid species.Note that sensitivity is partially restored after a 1 h polarity switch. (b) Sensitivity of the analysis of chemically sulfated sterols is not affected.The sulfated apolar lipid extract was infused using 0.005% (w/v) methylamine in pyridine/methanol (1:9, v/v), and analyzed for 3min bynegative ion mode FTMS analysis. Dashed grey lines indicate average values.Figure 2. Recommended sequence of MS analyses for globallipidome analysis using shotgun lipidomics. A standard samplepreparation routine includes two‐step lipid extraction and chemicalsulfation of the apolar lipid extract for the quantification of sterols.The recommended sequence of analyses is designed to avoid lossof sensitivity following injection of samples subjected to chemicalsulfation. Hence, the analysis of chemically sulfated lipid extractsshould be scheduled at the end of all other analyses for a particularset of samples and followed by positive ion mode analysis of thesubsequent set of samples.Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 Shotgun lipidomic analysis of chemically sulfated sterols 1619 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. www.ejlst.com[4] Adamovich, Y., Rousso‐Noori, L., Zwighaft, Z., Neufeld‐Cohen, A., et al., Circadian clocks and feeding time regulatethe oscillations and levels of hepatic triglycerides. Cell Metab.2014, 19, 319–330.[5] Tarasov, K., Stefanko, A., Casanovas, A., Surma, M. A.,et al., High‐content screening of yeast mutant libraries byshotgun lipidomics. Mol. Biosyst. 2014, 10, 1364–1376.[6] Han, X., Yang, K., Gross, R. W., Multi‐dimensional massspectrometry‐based shotgun lipidomics and novel strategiesfor lipidomic analyses. Mass Spectrom. Rev. 2012, 31, 134–178.[7] Higashi, T., Shimada, K., Derivatization of neutral steroidsto enhance their detection characteristics in liquid chroma-tography–mass spectrometry. Anal. Bioanal. Chem. 2004,378, 875–882.[8] Liebisch, G., Binder,M., Schifferer, R., Langmann, T., et al.,High throughput quantification of cholesterol and cholesterylester by electrospray ionization tandem mass spectrometry(ESI–MS/MS). Biochim. Biophys. Acta 2006, 1761, 121–128.[9] Sandhoff, R., Brugger, B., Jeckel, D., Lehmann, W. D.,Wieland, F. T., Determination of cholesterol at the lowpicomole level by nano‐electrospray ionization tandem massspectrometry. J. Lipid Res. 1999, 40, 126–132.[10] Klose, C., Surma, M. A., Gerl, M. J., Meyenhofer, F., et al.,Flexibility of a eukaryotic lipidome–insights from yeastlipidomics. PLoS ONE 2012, 7, e35063.[11] Husen, P., Tarasov, K., Katafiasz, M., Sokol, E., et al.,Analysis of lipid experiments (ALEX): A software frameworkfor analysis of high‐resolution shotgun lipidomics data. PLoSONE 2013, 8, e79736.1620 A. Casanovas et al. Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. www.ejlst.com

English

Shotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivity:Recommendation for large-scale global lipidome analysis

       University of Southern DenmarkShotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivityRecommendation for large-scale global lipidome analysisCasanovas, Albert; Hannibal-Bach, Hans Kristian; Jensen, Ole Nørregaard; Ejsing, Christer SPublished in:European Journal of Lipid Science and TechnologyDOI:10.1002/ejlt.201400451Publication date:2014Document version:Final published versionCitation for pulished version (APA):Casanovas, A., Hannibal-Bach, H. K., Jensen, O. N., & Ejsing, C. S. (2014). Shotgun lipidomic analysis ofchemically sulfated sterols compromises analytical sensitivity: Recommendation for large-scale global lipidomeanalysis. European Journal of Lipid Science and Technology, 116(12), 1618-1620.https://doi.org/10.1002/ejlt.201400451Go to publication entry in University of Southern Denmark's Research PortalTerms of useThis work is brought to you by the University of Southern Denmark.Unless otherwise specified it has been shared according to the terms for self-archiving.If no other license is stated, these terms apply:            • You may download this work for personal use only.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying this open access versionIf you believe that this document breaches copyright please contact us providing details and we will investigate your claim.Please direct all enquiries to puresupport@bib.sdu.dkDownload date: 22. Oct. 2025Letter to the EditorShotgun lipidomic analysis of chemically sulfated sterolscompromises analytical sensitivity: Recommendation forlarge‐scale global lipidome analysisAlbert Casanovas, Hans Kristian Hannibal‐Bach, Ole N. Jensen and Christer S. EjsingDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, DenmarkKeywords: Sensitivity / Shotgun lipidomics / Sterol sulfationReceived: September 12, 2014 / Accepted: September 26, 2014DOI: 10.1002/ejlt.201400451Shotgun lipidomics affords comprehensive and quantitativeanalysis of lipid species in cells and tissues at high‐throughput [1–5]. The methodology is based on directinfusion of lipid extracts by electrospray ionization (ESI)combined with tandem mass spectrometry (MS/MS) and/orhigh resolution Fourier transform mass spectrometry(FTMS) for identification and quantification of lipidspecies [6]. Shotgun lipidomics affords extensive lipidomecoverage by combining the analysis of lipid extracts in positiveand negative ion mode [1, 3]. Notably, sterols such ascholesterol and ergosterol exhibit low ionization efficiency inESI [7]. For this reason, chemical derivatization proceduresincluding acetylation [8] or sulfation [9] are commonlyimplemented to facilitate ionization, detection and quantifi-cation of sterols for global lipidome analysis [1–3, 10].In the course of large‐scale lipidomic analyses using anLTQ Orbitrap XL mass spectrometer (Thermo Scientific)equipped with a robotic nanoESI source TriVersa NanoMate(Advion Biosciences), we observed a pronounced decreasein the sensitivity of negative ion mode analysis followingrepeated injections of samples subjected to chemical sulfation.This decrease in sensitivity was observed only for negative ionmode analysis of lipid species in underivatized lipid extracts.No decrease in sensitivity was observed for negative ion modeanalysis of chemically sulfated sterols or for positive ion modeanalysis of lipid species in underivatized lipid extracts.To investigate the decrease in analytical sensitivity in furtherdetail we (i) spiked a yeast lysate with internal standards andexecuted two‐step lipid extraction as previously described [3]for the partition of apolar and polar lipids, (ii) subjected theapolar lipid extract (containing sterols) to chemical sulfation[2, 9], and (iii) infused the sulfated sample in batches of tenrepeated injections and monitored the intensity of chemicallysulfated sterols by negative ion mode FTMS. To monitor thedecrease in sensitivity we analyzed the underivatized polar lipidextract [containing phosphatidylinositol (PI) as well as otherpolar lipids [3]] by negative ion mode FTMS before and aftereach batch of ten repeated injections of the sulfated sample.This analysis showed a progressive loss of sensitivity fornegative ion mode analysis of lipid species in the under-ivatized polar lipid extract. To exemplify this effect, theintensity of PI 34:1, an abundant glycerophospholipid speciesin yeast [3, 10], was reduced more than 20‐fold after 30injections of the sulfated sample (Fig. 1a). More importantly,since an intensity threshold is routinely used in shotgunlipidomics data processing [11], the decrease in sensitivitytranslated into a decrease in the number of lipid speciesidentified and quantified (data not shown). Notably, theintensity of chemically sulfated sterols was unaffected(Fig. 1b). As we had previously observed that the sensitivityof positive ion mode analysis was not affected, we transientlyswitched the polarity to positive for 1 h and subsequentlyreverted it to evaluate whether the sensitivity of negative ionmode analysis was restored. This polarity switch partiallyrestored the sensitivity (>40%, in the case of PI 34:1) fornegative ion mode analysis of lipid species in the under-ivatized polar lipid extract (Fig. 1a). Based on these results,we recommend that global shotgun lipidomic experimentsemploying chemical sulfation of sterols use a specificsequence of MS analyses where the analysis of sulfatedsamples is scheduled at the end of all other analyses in ordernot to compromise analytical sensitivity (Fig. 2).In conclusion, here we: (i) demonstrate that shotgunlipidomic analysis of sample extracts subjected to chemicalsulfation can prompt a pronounced loss of sensitivity fornegative ion mode analysis of underivatized lipid extracts;Correspondence: Christer S. Ejsing, Department of Biochemistry andMolecular Biology, University of Southern Denmark, Campusvej 55, 5230Odense, DenmarkE‐mail: cse@bmb.sdu.dkFax: þ45 6550 2467Abbreviations: ESI, electrospray ionization;FTMS,Fourier transformmassspectrometry; MS/MS, tandem mass spectrometry; PI, phosphatidylinositol1618 Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.www.ejlst.comThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, whichpermits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercialand no modifications or adaptations are made.(ii) show that changing polarity is a way to overcome thisanalytical problem; and (iii) recommend that analysis ofchemically sulfated lipid extracts is scheduled at the end ofall other analyses in order not to compromise sensitivity anddata quality of large‐scale global lipidome analyses.We are grateful to Maria Carvalho for providing the protocolfor chemical sulfation of sterol lipids. This work was supported byLundbeckfonden (R44‐A4342, R54‐A5858, CSE), the DanishCouncil for Independent Research |Natural Sciences (10‐094213,AC; 09‐072484, CSE), and a Marie Curie Intra EuropeanFellowship (AC).The authors have declared no conflict of interest.References[1] Sampaio, J. L., Gerl, M. J., Klose, C., Ejsing, C. S., et al.,Membrane lipidome of an epithelial cell line. Proc. Natl.Acad. Sci. USA 2011, 108, 1903–1907.[2] Carvalho, M., Sampaio, J. L., Palm, W., Brankatschk, M.,et al., Effects of diet and development on the Drosophilalipidome. Mol. Syst. Biol. 2012, 8, 600.[3] Ejsing, C. S., Sampaio, J. L., Surendranath, V., Duchoslav,E., et al., Global analysis of the yeast lipidome by quantitativeshotgun mass spectrometry. Proc. Natl. Acad. Sci. USA 2009,106, 2136–2141.Figure 1. (a) Sensitivity of negative ion mode analysis of lipid species in the underivatized polar lipid extract is reduced after analysisof chemically sulfated sterols. The underivatized polar lipid extract was infused using 0.005% (w/v) methylamine in chloroform/methanol(1:5, v/v), and analyzed for 5min by negative ion mode FTMS analysis. The intensity of PI 34:1 is shown as a representative lipid species.Note that sensitivity is partially restored after a 1 h polarity switch. (b) Sensitivity of the analysis of chemically sulfated sterols is not affected.The sulfated apolar lipid extract was infused using 0.005% (w/v) methylamine in pyridine/methanol (1:9, v/v), and analyzed for 3min bynegative ion mode FTMS analysis. Dashed grey lines indicate average values.Figure 2. Recommended sequence of MS analyses for globallipidome analysis using shotgun lipidomics. A standard samplepreparation routine includes two‐step lipid extraction and chemicalsulfation of the apolar lipid extract for the quantification of sterols.The recommended sequence of analyses is designed to avoid lossof sensitivity following injection of samples subjected to chemicalsulfation. Hence, the analysis of chemically sulfated lipid extractsshould be scheduled at the end of all other analyses for a particularset of samples and followed by positive ion mode analysis of thesubsequent set of samples.Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 Shotgun lipidomic analysis of chemically sulfated sterols 1619 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. www.ejlst.com[4] Adamovich, Y., Rousso‐Noori, L., Zwighaft, Z., Neufeld‐Cohen, A., et al., Circadian clocks and feeding time regulatethe oscillations and levels of hepatic triglycerides. Cell Metab.2014, 19, 319–330.[5] Tarasov, K., Stefanko, A., Casanovas, A., Surma, M. A.,et al., High‐content screening of yeast mutant libraries byshotgun lipidomics. Mol. Biosyst. 2014, 10, 1364–1376.[6] Han, X., Yang, K., Gross, R. W., Multi‐dimensional massspectrometry‐based shotgun lipidomics and novel strategiesfor lipidomic analyses. Mass Spectrom. Rev. 2012, 31, 134–178.[7] Higashi, T., Shimada, K., Derivatization of neutral steroidsto enhance their detection characteristics in liquid chroma-tography–mass spectrometry. Anal. Bioanal. Chem. 2004,378, 875–882.[8] Liebisch, G., Binder,M., Schifferer, R., Langmann, T., et al.,High throughput quantification of cholesterol and cholesterylester by electrospray ionization tandem mass spectrometry(ESI–MS/MS). Biochim. Biophys. Acta 2006, 1761, 121–128.[9] Sandhoff, R., Brugger, B., Jeckel, D., Lehmann, W. D.,Wieland, F. T., Determination of cholesterol at the lowpicomole level by nano‐electrospray ionization tandem massspectrometry. J. Lipid Res. 1999, 40, 126–132.[10] Klose, C., Surma, M. A., Gerl, M. J., Meyenhofer, F., et al.,Flexibility of a eukaryotic lipidome–insights from yeastlipidomics. PLoS ONE 2012, 7, e35063.[11] Husen, P., Tarasov, K., Katafiasz, M., Sokol, E., et al.,Analysis of lipid experiments (ALEX): A software frameworkfor analysis of high‐resolution shotgun lipidomics data. PLoSONE 2013, 8, e79736.1620 A. Casanovas et al. Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. www.ejlst.com

       University of Southern DenmarkShotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivityRecommendation for large-scale global lipidome analysisCasanovas, Albert; Hannibal-Bach, Hans Kristian; Jensen, Ole Nørregaard; Ejsing, Christer SPublished in:European Journal of Lipid Science and TechnologyDOI:10.1002/ejlt.201400451Publication date:2014Document version:Final published versionCitation for pulished version (APA):Casanovas, A., Hannibal-Bach, H. K., Jensen, O. N., & Ejsing, C. S. (2014). Shotgun lipidomic analysis ofchemically sulfated sterols compromises analytical sensitivity: Recommendation for large-scale global lipidomeanalysis. European Journal of Lipid Science and Technology, 116(12), 1618-1620.https://doi.org/10.1002/ejlt.201400451Go to publication entry in University of Southern Denmark's Research PortalTerms of useThis work is brought to you by the University of Southern Denmark.Unless otherwise specified it has been shared according to the terms for self-archiving.If no other license is stated, these terms apply:            • You may download this work for personal use only.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying this open access versionIf you believe that this document breaches copyright please contact us providing details and we will investigate your claim.Please direct all enquiries to puresupport@bib.sdu.dkDownload date: 25. Oct. 2025Letter to the EditorShotgun lipidomic analysis of chemically sulfated sterolscompromises analytical sensitivity: Recommendation forlarge‐scale global lipidome analysisAlbert Casanovas, Hans Kristian Hannibal‐Bach, Ole N. Jensen and Christer S. EjsingDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, DenmarkKeywords: Sensitivity / Shotgun lipidomics / Sterol sulfationReceived: September 12, 2014 / Accepted: September 26, 2014DOI: 10.1002/ejlt.201400451Shotgun lipidomics affords comprehensive and quantitativeanalysis of lipid species in cells and tissues at high‐throughput [1–5]. The methodology is based on directinfusion of lipid extracts by electrospray ionization (ESI)combined with tandem mass spectrometry (MS/MS) and/orhigh resolution Fourier transform mass spectrometry(FTMS) for identification and quantification of lipidspecies [6]. Shotgun lipidomics affords extensive lipidomecoverage by combining the analysis of lipid extracts in positiveand negative ion mode [1, 3]. Notably, sterols such ascholesterol and ergosterol exhibit low ionization efficiency inESI [7]. For this reason, chemical derivatization proceduresincluding acetylation [8] or sulfation [9] are commonlyimplemented to facilitate ionization, detection and quantifi-cation of sterols for global lipidome analysis [1–3, 10].In the course of large‐scale lipidomic analyses using anLTQ Orbitrap XL mass spectrometer (Thermo Scientific)equipped with a robotic nanoESI source TriVersa NanoMate(Advion Biosciences), we observed a pronounced decreasein the sensitivity of negative ion mode analysis followingrepeated injections of samples subjected to chemical sulfation.This decrease in sensitivity was observed only for negative ionmode analysis of lipid species in underivatized lipid extracts.No decrease in sensitivity was observed for negative ion modeanalysis of chemically sulfated sterols or for positive ion modeanalysis of lipid species in underivatized lipid extracts.To investigate the decrease in analytical sensitivity in furtherdetail we (i) spiked a yeast lysate with internal standards andexecuted two‐step lipid extraction as previously described [3]for the partition of apolar and polar lipids, (ii) subjected theapolar lipid extract (containing sterols) to chemical sulfation[2, 9], and (iii) infused the sulfated sample in batches of tenrepeated injections and monitored the intensity of chemicallysulfated sterols by negative ion mode FTMS. To monitor thedecrease in sensitivity we analyzed the underivatized polar lipidextract [containing phosphatidylinositol (PI) as well as otherpolar lipids [3]] by negative ion mode FTMS before and aftereach batch of ten repeated injections of the sulfated sample.This analysis showed a progressive loss of sensitivity fornegative ion mode analysis of lipid species in the under-ivatized polar lipid extract. To exemplify this effect, theintensity of PI 34:1, an abundant glycerophospholipid speciesin yeast [3, 10], was reduced more than 20‐fold after 30injections of the sulfated sample (Fig. 1a). More importantly,since an intensity threshold is routinely used in shotgunlipidomics data processing [11], the decrease in sensitivitytranslated into a decrease in the number of lipid speciesidentified and quantified (data not shown). Notably, theintensity of chemically sulfated sterols was unaffected(Fig. 1b). As we had previously observed that the sensitivityof positive ion mode analysis was not affected, we transientlyswitched the polarity to positive for 1 h and subsequentlyreverted it to evaluate whether the sensitivity of negative ionmode analysis was restored. This polarity switch partiallyrestored the sensitivity (>40%, in the case of PI 34:1) fornegative ion mode analysis of lipid species in the under-ivatized polar lipid extract (Fig. 1a). Based on these results,we recommend that global shotgun lipidomic experimentsemploying chemical sulfation of sterols use a specificsequence of MS analyses where the analysis of sulfatedsamples is scheduled at the end of all other analyses in ordernot to compromise analytical sensitivity (Fig. 2).In conclusion, here we: (i) demonstrate that shotgunlipidomic analysis of sample extracts subjected to chemicalsulfation can prompt a pronounced loss of sensitivity fornegative ion mode analysis of underivatized lipid extracts;Correspondence: Christer S. Ejsing, Department of Biochemistry andMolecular Biology, University of Southern Denmark, Campusvej 55, 5230Odense, DenmarkE‐mail: cse@bmb.sdu.dkFax: þ45 6550 2467Abbreviations: ESI, electrospray ionization;FTMS,Fourier transformmassspectrometry; MS/MS, tandem mass spectrometry; PI, phosphatidylinositol1618 Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.www.ejlst.comThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, whichpermits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercialand no modifications or adaptations are made.(ii) show that changing polarity is a way to overcome thisanalytical problem; and (iii) recommend that analysis ofchemically sulfated lipid extracts is scheduled at the end ofall other analyses in order not to compromise sensitivity anddata quality of large‐scale global lipidome analyses.We are grateful to Maria Carvalho for providing the protocolfor chemical sulfation of sterol lipids. This work was supported byLundbeckfonden (R44‐A4342, R54‐A5858, CSE), the DanishCouncil for Independent Research |Natural Sciences (10‐094213,AC; 09‐072484, CSE), and a Marie Curie Intra EuropeanFellowship (AC).The authors have declared no conflict of interest.References[1] Sampaio, J. L., Gerl, M. J., Klose, C., Ejsing, C. S., et al.,Membrane lipidome of an epithelial cell line. Proc. Natl.Acad. Sci. USA 2011, 108, 1903–1907.[2] Carvalho, M., Sampaio, J. L., Palm, W., Brankatschk, M.,et al., Effects of diet and development on the Drosophilalipidome. Mol. Syst. Biol. 2012, 8, 600.[3] Ejsing, C. S., Sampaio, J. L., Surendranath, V., Duchoslav,E., et al., Global analysis of the yeast lipidome by quantitativeshotgun mass spectrometry. Proc. Natl. Acad. Sci. USA 2009,106, 2136–2141.Figure 1. (a) Sensitivity of negative ion mode analysis of lipid species in the underivatized polar lipid extract is reduced after analysisof chemically sulfated sterols. The underivatized polar lipid extract was infused using 0.005% (w/v) methylamine in chloroform/methanol(1:5, v/v), and analyzed for 5min by negative ion mode FTMS analysis. The intensity of PI 34:1 is shown as a representative lipid species.Note that sensitivity is partially restored after a 1 h polarity switch. (b) Sensitivity of the analysis of chemically sulfated sterols is not affected.The sulfated apolar lipid extract was infused using 0.005% (w/v) methylamine in pyridine/methanol (1:9, v/v), and analyzed for 3min bynegative ion mode FTMS analysis. Dashed grey lines indicate average values.Figure 2. Recommended sequence of MS analyses for globallipidome analysis using shotgun lipidomics. A standard samplepreparation routine includes two‐step lipid extraction and chemicalsulfation of the apolar lipid extract for the quantification of sterols.The recommended sequence of analyses is designed to avoid lossof sensitivity following injection of samples subjected to chemicalsulfation. Hence, the analysis of chemically sulfated lipid extractsshould be scheduled at the end of all other analyses for a particularset of samples and followed by positive ion mode analysis of thesubsequent set of samples.Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 Shotgun lipidomic analysis of chemically sulfated sterols 1619 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. www.ejlst.com[4] Adamovich, Y., Rousso‐Noori, L., Zwighaft, Z., Neufeld‐Cohen, A., et al., Circadian clocks and feeding time regulatethe oscillations and levels of hepatic triglycerides. Cell Metab.2014, 19, 319–330.[5] Tarasov, K., Stefanko, A., Casanovas, A., Surma, M. A.,et al., High‐content screening of yeast mutant libraries byshotgun lipidomics. Mol. Biosyst. 2014, 10, 1364–1376.[6] Han, X., Yang, K., Gross, R. W., Multi‐dimensional massspectrometry‐based shotgun lipidomics and novel strategiesfor lipidomic analyses. Mass Spectrom. Rev. 2012, 31, 134–178.[7] Higashi, T., Shimada, K., Derivatization of neutral steroidsto enhance their detection characteristics in liquid chroma-tography–mass spectrometry. Anal. Bioanal. Chem. 2004,378, 875–882.[8] Liebisch, G., Binder,M., Schifferer, R., Langmann, T., et al.,High throughput quantification of cholesterol and cholesterylester by electrospray ionization tandem mass spectrometry(ESI–MS/MS). Biochim. Biophys. Acta 2006, 1761, 121–128.[9] Sandhoff, R., Brugger, B., Jeckel, D., Lehmann, W. D.,Wieland, F. T., Determination of cholesterol at the lowpicomole level by nano‐electrospray ionization tandem massspectrometry. J. Lipid Res. 1999, 40, 126–132.[10] Klose, C., Surma, M. A., Gerl, M. J., Meyenhofer, F., et al.,Flexibility of a eukaryotic lipidome–insights from yeastlipidomics. PLoS ONE 2012, 7, e35063.[11] Husen, P., Tarasov, K., Katafiasz, M., Sokol, E., et al.,Analysis of lipid experiments (ALEX): A software frameworkfor analysis of high‐resolution shotgun lipidomics data. PLoSONE 2013, 8, e79736.1620 A. Casanovas et al. Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620 2015 The Authors. European Journal of Lipid Science and TechnologyPublished by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. www.ejlst.com

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Shotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivity:Recommendation for large-scale global lipidome analysis

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Syddansk Universitets Forskerportal

Syddansk Universitets Forskerportal

Syddansk Universitets Forskerportal