Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers. Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers. Glycoglycerolipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol [DGDG], and sulfolipid [SQDG]) are the most abundant membrane lipids in chloroplasts of plants (1Joyard J. Block M.A. Malhèrbe A. Maréchal E. Douce R. Origin and synthesis of galactolipid and sulfolipid head groups.in: Moore T.S.J. Lipid Metabolism In Plants. CRC Press, Boca Raton (FL)1994: 231-258Google Scholar, 2Benning C. Ohta H. Three enzyme systems for galactoglycerolipid biosynthesis are coordinately regulated in plants.J. Biol. Chem. 2005; 280: 2397-2400Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Different lipid classes, however, are prevalent in extraplastidial membranes. Phosphoglycerolipids (e.g., phosphatidylcholine and phosphatidylethanolamine) are abundant constituents of the plasma membrane, the tonoplast, and the endoplasmic reticulum. Furthermore, sphingolipids, in particular glucosylceramides and glycosylinositolphosphoceramides, are present in extraplastidial membranes of plants (3Markham J.E. Jaworski J.G. Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2007; 21: 1304-1314Crossref PubMed Scopus (201) Google Scholar, 4Markham J.E. Li J. Cahoon E.B. Jaworski J.G. Separation and identification of major plant sphingolipid classes from leaves.J. Biol. Chem. 2006; 281: 22684-22694Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In addition to glycerolipids and sphingolipids, sterol lipids represent the third membrane lipid class in plants. In contrast to animals and yeast, where cholesterol and ergosterol, respectively, are prevalent, and represent the most abundant sterols in plants and of Biol. PubMed Scopus Google Scholar). The for provides the for in the of plant and in the PubMed Scopus Google Scholar). Free sterols with a are in the plasma membrane and other extraplastidial membranes of plant In classes of conjugated sterols are in sterol sterol and acylated sterol amounts of in contrast to the other sterol lipid classes, are to the of the The sterol of J. PubMed Scopus Google Scholar, E. Sterol of membranes and of in sterol PubMed Scopus Google Scholar). are the of lipid and amounts of and were in and of from or from a respectively, the of sterols A. A. H. sterol synthesis in plants is by enzyme from the and Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. of identification of a sterol in 2007; PubMed Scopus Google Scholar). in and is that of sterols is crucial for the of the in plant membranes A. A. A. H. of the sterol in plant sterol and PubMed Scopus Google Scholar). a in of the of the sterol C. of free sterols, and membrane PubMed Google Scholar, A. J. 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Li in in Arabidopsis and in PubMed Scopus Google Scholar, H. A. Lipid in plant plasma is to the J. PubMed Scopus Google Scholar). strategy provides the sterol of sterol lipid classes the of molecular species of and Furthermore, systems are to the and and the sterol lipids and In method is and from a of the of mass in the of and for the identification and quantification of lipids The of 2005; PubMed Scopus Google Scholar). Phosphoglycerolipids and can be in lipid by coupled with mass spectrometry R. Li Li H. C. membrane lipids in plant of in lipid changes in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Li R. R. of species from of Arabidopsis and a 2006; PubMed Scopus Google Scholar). In the of the molecular lipid species are by the after the lipids are quantified fragmentation or The of lipids coupled with a mass was for the quantification of plant sphingolipids (3Markham J.E. Jaworski J.G. Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2007; 21: 1304-1314Crossref PubMed Scopus (201) Google Scholar, 4Markham J.E. Li J. Cahoon E.B. Jaworski J.G. Separation and identification of major plant sphingolipid classes from leaves.J. Biol. Chem. 2006; 281: 22684-22694Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). by the sphingolipids are quantified of the mass after i.e., (Q-TOF MS) was to the of lipids sphingolipids, and diacylglycerol E. A. of the by mass A. PubMed Scopus Google Scholar). of were for the of and sterols from animals or by or J. J. spectrometry fragmentation for the identification of Lipid 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, R. quantification of cholesterol and by electrospray ionization tandem mass spectrometry 2006; PubMed Scopus Google Scholar, A. 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C. and of the Arabidopsis in to the PubMed Scopus Google was obtained from the Arabidopsis The to is as the and were used as internal for the quantification of for the quantification of were and are where the of and the of in the The used as internal were as synthesis of and 2005; PubMed Scopus Google and of were in of and to a of of of and of in of The was for the was with of and after was The was a of in of and with The was with and the were with and The were by or by and quantified by sterol from and were with in in the presence of as J. J. the of glycerolipids and in Arabidopsis 2006; PubMed Scopus Google Scholar). of the and sterol was by Q-TOF MS/MS sterol lipids were with and were as from and were quantified by The sterol of and were and quantified by The lipid of of and of and of and of of of and of Arabidopsis leaves or roots were to a in liquid lipids were with of and of a of and as The internal was The was and to The was with a with of were and the were The was a of and were by were to the in and lipids and were with of The and was obtained by with of The lipid or were and in of Q-TOF were derivatized with to Q-TOF were from leaves of and and by as The and and the and were was used for Q-TOF and the other was used for quantification by and GC lipids and were by of with a by to the in and with and the of were with ammonium and for The were in C. and of Arabidopsis in the lipid digalactosyl PubMed Scopus Google and lipids were from the with GC were in of in for and with H. of the in results in sterol PubMed Scopus Google Scholar). and were in of for the addition of of the sterols were with of were derivatized with of to GC was in and the were in GC was with GC with ionization sterols were a a of to for and to were by the and Free sterols were derivatized with the for diacylglycerol Quantification of diacylglycerol molecular species in by electrospray ionization mass spectrometry after Chem. 2007; PubMed Scopus Google Scholar). lipids after were in of chloride. of and of were and the was for with the The sterols were with of and of sterols were for by Q-TOF MS/MS the synthesis of of was to of Scholar). The was to and the addition of of the was to with was and the was the were were with and by were the Q-TOF mass Mass Q-TOF in the positive mode nanospray with Sterol lipids were to the mass in ammonium R. Li Li H. C. membrane lipids in plant of in lipid changes in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a of The of the in the was a of The was and the was to fragmentation were after MS/MS MS/MS the was in the and was the mass of the sterol Q-TOF MS/MS were for and the of from the were for MS/MS for and were and sterol lipid classes were quantified by Q-TOF MS/MS were of was for obtained by were Free sterols were quantified the of the of the to the of a of sterols in MS/MS than of a of sterols and and was quantified by GC and by Q-TOF a for sterols was were quantified by the of of internal were used to for the in for with or fragmentation of of the sterol Therefore, a of and was GC and Q-TOF to the the Q-TOF MS/MS was to be for than for by a of was used for the during Q-TOF MS/MS of with sterol a fragmentation with a with a sterol a Quantification of the and in a of and by Q-TOF MS/MS revealed that the for with and sterol were no was of all molecular sterol species and of the MS/MS from the presence of were the mass and the for the molecular species of all sterol classes are in was in all where a molecular species that in the sterol or in the as in a E. J. R. C. A. identification and quantification of molecular species by Chem. 2006; PubMed Scopus Google Scholar). was for the of used for lipid class to for the of ionization the of the R. of membrane lipids the by ionization tandem mass A. PubMed Scopus Google Scholar). The of molecular species were in of sterol lipids Sterol lipids were the Q-TOF mass nanospray were in the positive mode Free sterols from Arabidopsis leaves after and as and were used as internal standards. Sterol from Arabidopsis leaves after The internal were and Sterol internal were and sterol and were the internal of sterol and acylated sterol Q-TOF MS/MS of of the ammonium of a sterol The of the as the internal a of to the sterol and the presence of Q-TOF MS/MS of that fragmentation of with sterol to the of the sterol of with sterol internal to the of a to the of free sterols from and were by Q-TOF of the of the of the after with internal and were by Q-TOF of the of the of the after with chloride. in a internal MS/MS a or a Q-TOF mass represent a and method for the identification and quantification of lipids R. Li Li H. C. membrane lipids in plant of in lipid changes in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. of the by mass A. PubMed Scopus Google Scholar, R. of membrane lipids the by ionization tandem mass A. 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PubMed Scopus Google Scholar). major of can from in the ionization of from a is that the of a is a major for the ionization in the Therefore, can from during The four sterol lipid classes present in plants in with and and are the of a was from a Arabidopsis lipid and for sterol lipids were in the and MS/MS of the nanospray Q-TOF mass was for and and were Therefore, the lipid was and lipids and or lipids and were in a of ionization for all sterol lipid classes, in particular for sterol and was for all of lipids in ammonium in the ionization of most sterol lipids and as ammonium Free sterols, however, were were and the of the of to ionization during fragmentation can a can be in a or Free and were quantified in MS/MS after to of or or to R. quantification of cholesterol and by electrospray ionization tandem mass spectrometry 2006; PubMed Scopus Google Scholar, A. 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Full Text Full Text PDF PubMed Scopus Google Scholar, R. of membrane lipids the by ionization tandem mass A. PubMed Scopus Google Scholar). sterols are from plants and can as internal standards. and were used as internal for free sterol of are to in particular a with are liquid chromatography with a revealed that of were the of of the Separation of lipids by chromatography and by electrospray ionization mass Lipid Full Text Full Text PDF PubMed Scopus Google Scholar). nanospray MS/MS with a Q-TOF that the for and were for of with of internal were The of and were for the measurement of with and the quantification of and were a from was and were used as internal for The for lipid quantification MS/MS is the of the in the the fragmentation in the and the identification and quantification of a in the mass or in the mass The was to fragmentation of conjugated sterols the Furthermore, were for for the The are in and a positive that in the of the sterol as and in the of the of of ammonium of to the of the The of the was used for fragmentation of to that of the sterol of obtained in the mode with obtained in the MS/MS mode that fragmentation of a for the the was and a of was of particular the internal sterol plant of for the quantification of sphingolipid classes (3Markham J.E. Jaworski J.G. Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2007; 21: 1304-1314Crossref PubMed Scopus (201) Google Scholar). was for the quantification of by of a to for the of to the fragmentation of the was or more were present in the sterol The fragmentation of sterol in the of a for the sterol of with a sterol however, to the of a for the Therefore, the and were for quantification of with or sterol The of was for sterol lipid the standards. for in the nanospray of the the of sterol lipid was to a internal of the lipid was The of the were to the of the that the were a of to of for and and than of for for for all four lipid classes were to be than and were to be the most abundant sterols in Arabidopsis A. Li in in Arabidopsis and in PubMed Scopus Google Scholar, R. Arabidopsis in sterol by a from J. PubMed Scopus Google Scholar). and are for molecular species of sterol lipids the Q-TOF and MS/MS Therefore, sterols were for a to the sterol lipid and are conjugated sterol lipids of sterols after were quantified and a of molecular species were by and MS/MS with from to and to all of were in or of a of molecular species was for MS/MS to in the during or in plant species The most abundant in Arabidopsis leaves were and a of and The molecular species of was by with amounts of and the of all sterol lipid classes for and the used for quantification are in of sterol from molecular and were by Q-TOF of the of the of the with internal and were by Q-TOF of the of the of the with in a of sterol from molecular and were by Q-TOF of the of the of the with internal and were by Q-TOF of the of the of the with in a of acylated sterol from sterol molecular and were by Q-TOF of the of the of the with internal and were by Q-TOF of the of the of the with in a internal internal internal quantification of sterol lipids from Arabidopsis leaves was used to the quantification results by Q-TOF a lipid from Arabidopsis leaves internal was and and and sterols was for Q-TOF was used for by and and quantification by GC after and a comparison of the sterol lipid as by and Q-TOF The in the and amounts of sterol lipids from Arabidopsis represent the sterol lipid and are and represent a lipid of the most for of membrane lipid in plants. the changes in the of and during the the amounts of sterol lipids are Therefore, the and of sterol lipids were by Q-TOF MS/MS after were from leaves or roots of Arabidopsis plants with or Furthermore, lipids were from the Arabidopsis of the are to a in a that from the to the E. J. C. and of the Arabidopsis in to the PubMed Scopus Google Scholar, C. J. of Arabidopsis in of PubMed Scopus Google Scholar). the sterol lipid by Q-TOF MS/MS in leaves of the and the and in leaves and roots of plants in the presence or of a in the amounts of and in be in leaves of the with in the The of was in roots during the in roots was the the of was in leaves of phosphate-deprived was and in were in with obtained from H. A. Lipid in plant plasma is to the J. PubMed Scopus Google Scholar). The amounts of were in phosphate-deprived leaves and phosphate-deprived roots of the Furthermore, leaves of Arabidopsis plants a than was during The amounts of and in the sterol lipid classes were of the changes were to be the were in of and during The of in the and in the leaves of plants in the of was the the amounts of in were Furthermore, a of were in of and the amounts of were during In the amounts of were during with a in to the changes in of In was in with a of that was a from to of sterol and acylated sterol by Q-TOF Sterol acylated sterol Sterol lipids were from leaves of and plants and from leaves and roots of plants with or as of were by a that are from of the or and than are by to The of mass the for the of to the identification and quantification of plant In contrast to and of are for the of membrane and the of a comprehensive strategy for the identification and quantification of the molecular species of all sterol lipid classes in plants Q-TOF Sterol lipid classes in plants with to the ionization and fragmentation in and MS/MS to the molecular species of all four sterol lipid classes by MS/MS of a lipid were The ionization of the sterol lipids was after ionization of was of ionization and the for of a in of was the presence or of a in the with were more to than the and were quantified to the or internal standards. ionization of and was fragmentation were to the presence or of a in the sterol The of the sterol were for with of was to for the in fragmentation the of the sterol of with sterol was than that of with The of with sterol was for and a to the acylated head was In the present sterols and sterols of the or were for and sterol lipids were that the a ionization and In with A. Li in in Arabidopsis and in PubMed Scopus Google Scholar, R. Arabidopsis in sterol by a from J. PubMed Scopus Google and were to be the sterol in Arabidopsis as by Q-TOF and were and are with the molecular lipids or to and be in MS/MS and were prevalent in of leaves and roots from Arabidopsis roots a of was from are to amounts of The of in that a of in was from the the endoplasmic reticulum. In contrast to are prevalent in to of all of In amounts of and are in The of sterol lipids obtained by Q-TOF MS/MS quantification were to the sterol lipid amounts by a of and the of and in Arabidopsis leaves were as and R. Arabidopsis in sterol by a from J. PubMed Scopus Google Scholar). to and and for and were in a A. A. A. H. of the sterol in plant sterol and PubMed Scopus Google Scholar). that the for Arabidopsis leaves is and for and respectively, is in the as the of and by Q-TOF MS/MS in The of and in leaves to the other sterol lipids were the and A. Li in in Arabidopsis and in PubMed Scopus Google Scholar). are to from Q-TOF MS/MS i.e., and The sterol of Arabidopsis leaves were as and and and for and in and R. Arabidopsis in sterol by a from J. PubMed Scopus Google Scholar). are in with by Q-TOF and and and for and in and and be by Q-TOF is to a in the and and a in provides the to during C. Ohta H. Three enzyme systems for galactoglycerolipid biosynthesis are coordinately regulated in plants.J. Biol. Chem. 2005; 280: 2397-2400Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). and represent and are to extraplastidial in particular to the plasma changes in plasma membrane lipid were for roots H. A. Lipid in plant plasma is to the J. PubMed Scopus Google Scholar, H. C. The plasma membrane and the as major for and of in the plasma Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). was in the of and to the plasma membrane of sterol lipids in Arabidopsis leaves and roots by Q-TOF MS/MS after were in with the results obtained with In leaves of the and the plants of and were in were changes in In the in leaves and roots was during The was during in as represent plasma membrane lipid H. A. Lipid in plant plasma is to the J. PubMed Scopus Google Scholar, H. C. The plasma membrane and the as major for and of in the plasma Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the in leaves of Arabidopsis plants was with that in plants that the of in plants the and in Scopus Google Scholar). In the of with plant and during and abiotic is that the of in of is in to the of from the plasma membrane and a of membrane lipid the of of or after to a than a to The of and were more to changes in than the sterol more or of with a of in and was in the and in leaves of In of the of was can be by synthesis of with and Q-TOF with MS/MS provides a and sensitive method for the quantification of sterol for all sterol lipid classes were to be than and of was to all molecular species of sterol lipids in Arabidopsis of the Q-TOF method is the that the molecular species of and can be Furthermore, method can be to sterol lipids in and in other plant species or by molecular species to the MS/MS are to a in with is in to the that ionization is of the of during and to the that a is In all sterol lipid by Q-TOF MS/MS be with a lipid from a Arabidopsis The for quantification of the molecular species of sterol lipids was in the of in of by Q-TOF a of a of to is to the fragmentation for quantification of all sterol Therefore, the for quantification of molecular sterol lipid species is In the of sterol lipid for quantification by and GC or by with in the Furthermore, the of for Q-TOF MS/MS of all four sterol lipid classes is with for the method is far more sensitive than of sterol lipid of the Q-TOF method is the that is as is of The sterol lipid quantification method was a Q-TOF with a nanospray Quantification was MS/MS The for the were from the of all (e.g., for or (e.g., for the Q-TOF strategy of sterol lipid quantification can be to other tandem mass with a nanospray or The of for during Q-TOF and of for with acylated sterol digalactosyl diacylglycerol free sterol tandem mass spectrometry sterol sterol sulfolipid