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A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversed-phase LC-MS or LC-tandem mass spectrometry using an Atlantis dC18 capillary column, and/or nanospray MS, to fully characterize the individual lipids. The methodology is applied here for the analysis of human apolipoprotein B-associated lipids. This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. The methods described should be easily adaptable to most modern MS instrumentation. A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversed-phase LC-MS or LC-tandem mass spectrometry using an Atlantis dC18 capillary column, and/or nanospray MS, to fully characterize the individual lipids. The methodology is applied here for the analysis of human apolipoprotein B-associated lipids. This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. The methods described should be easily adaptable to most modern MS instrumentation. A large variety of methods have been published for the separation of lipids, either by TLC or by LC; the methods have usually been described for the analysis of specific classes of compounds (www.cyberlipid.org). MS methods for the characterization of lipid mixtures have also been published in recent years, most of them centered on the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and electrospray ionization (ESI) MS (1Murphy R.C. Fiedler J. Hevko J. Analysis of nonvolatile lipids by mass spectrometry.Chem. Rev. 2001; 101: 479-526Crossref PubMed Scopus (235) Google Scholar) (in addition to the references cited in the text, important Web-based sources of information were www.cyberlipid.org, www.lipidlibrary.co.uk, and www.lipidmaps.org). Sophisticated methods like the characterization of complex glycolipids directly from TLC plates by vibrationally cooled MALDI Fourier transform-ion cyclotron resonance MS (2Ivleva V.B. Elkin Y.N. Budnik B.A. Moyer S.C. O'Connor P.B. Costello C.E. Coupling thin-layer chromatography with vibrational cooling matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for the analysis of ganglioside mixtures.Anal. Chem. 2004; 76: 6484-6491Crossref PubMed Scopus (96) Google Scholar) require instrumentation that is not yet widely available. A variety of elegant nanospray MS methods have been described (3Bruägger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (734) Google Scholar, 4Han X. Yang J. Cheng H. Ye H. Gross R.W. Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Crossref PubMed Scopus (179) Google Scholar, 5Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (918) Google Scholar) that are generally a good choice for the characterization of lipids, but they may not be fully capable of both qualitative and quantitative analysis of highly complex mixtures. LC-MS offers possibilities for a better determination of minor compounds whose signals might otherwise be suppressed. It also allows for an additional level of characterization of components based on their chromatographic behavior as well as the MS results. Existing HPLC methods for the separation of lipids are limited, however, in that they either target only selected classes or are not compatible with subsequent MS. Pulfer and Murphy (6Pulfer M. Murphy R.C. Electrospray mass spectrometry of phospholipids.Mass Spectrom. Rev. 2003; 22: 332-364Crossref PubMed Scopus (733) Google Scholar) suggested that, for a complete separation of lipids, normal- and reversed-phase chromatography should be combined. Because of its high sensitivity and the additional information it provides, MS is widely recognized as a superior detection method compared with the classic methods of ultraviolet or light scattering. We demonstrate here a simple, robust, and reproducible methodology for lipid analysis, which has been achieved by adapting to LC-MS several separation systems described in the literature for the thin-layer and liquid chromatography of lipids. After an optional initial cleanup and prefractionation on Silica 60, we use normal-phase HPLC-MS for class separation first, then an optional reversed-phase LC-MS or LC-tandem mass spectrometry (MS/MS) system for further analysis. Application of the methodology for the analysis of human LDL lipids is demonstrated here. We compared the lipids associated with normal apolipoprotein B-100 (B100)-containing LDL with those associated with mutant apolipoprotein B-67 (B67)-containing lipoproteins, which are found in certain cases of familial hypobetalipoproteinemia (7Welty F.K. Hubl S.T. Pierotti V.R. Young S.G. A truncated species of apolipoprotein B (B67) in a kindred with familial hypobetalipoproteinemia.J. Clin. Invest. 1991; 87: 1748-1754Crossref PubMed Scopus (31) Google Scholar). Lipid standards were obtained from Avanti (Alabaster, AL), Matreya (Pleasant Gap, PA), or Sigma (St. Louis, MO) and were prepared for use in this study as 1 mg/ml total lipid stock solutions. Whereas the simple acylglycerols (monoacylglycerol, diacylglycerol, and triacylglycerol), cholesterol, cholesteryl esters, free fatty acids, and fatty acid methyl ester standards and the glycolipids (prepared as 1 mg/ml stock solutions) each consisted of only one species, typically the oleoyl (C18:1) derivatives, the phospholipid standards each contained multiple components. Lipid structures are provided in the Supplemental Figure. The solvents used were HPLC-grade. Most experiments were carried out using a Hewlett-Packard (now Agilent) 1090 HPLC system coupled to a Waters/Micromass (Beverly, MA) Quattro II triple quadrupole (QQQ) mass spectrometer. Both were controlled with Waters/Micromass MassLynx 3.4 software, which was also used for used were quadrupole time-of-flight mass both controlled by and this was also used for of the was with a nanospray and the was to a that was controlled by MassLynx from a normal individual was isolated by A of was isolated and at lipoproteins, and LDL from a patient to were isolated by were to and A method of total lipid and J. Biochem. PubMed Scopus Google Scholar). The human samples were with and the of the Lipid standards and the isolated and LDL lipids were Silica and were with methyl followed by with or PA), were The samples were a of the from the or the a of were on a YMC PVA-Sil column MA) on the Quattro II with separation, and detection in both and class was as lipid standards for LC-MS methods were the determination of lipids, the solvents were in in or a of A was for followed by a to which was for and of The only of the to the mass spectrometer. the in and was to and the was for lipids and for lipids. the determination of the lipids, the solvents were in and in or in The method consisted of of a to which was for of solvents of and A of of in was with a to the the mass to ionization were a of further obtained from the normal-phase column be further by reversed-phase using a Atlantis dC18 capillary column with either the Quattro system described or with a system to an mass spectrometer. were either with a to or a of standards were the separation of both the and the solvents and were in and in The method consisted of for a to and for the mass was typically to and to in and to and in LC-MS for in were at a of and a of with mass in at and MS was on either of mass spectrometer. were prepared from using a were on the using MS and in the or as described by (3Bruägger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (734) Google Scholar). were for in the and for and in the as well as an additional mass for in the selected were also obtained with the MS system using MS and in the and was based on the on the PVA-Sil the experiments with biological obtained for standards on the were used for of the standards contained a typically class for each for differences in were for classes usually was used for and to the was used for and The were based on or of the at the The were not for in ionization a for use as standards and a in this study was to a robust methodology that be used with simple as well as instrumentation and that is for the analysis of complex mixtures that are to by nanospray MS. for biological samples or or in the of of lipids in the of large of lipids we used an initial separation on Silica the separation we the of separation by classes by normal-phase LC-MS the information for a quantitation of minor as well as a level of information the molecular that might be to the in from this normal-phase column then be further by reversed-phase LC-MS and/or by nanospray MS. Lipid standards and glycolipids have been on the of by from Silica with and as cholesteryl esters, and were found to be well but compounds of were in in both of the large of that to be but not reproducible were used only for samples of for or addition of to the not the the most the be with with or and were selected for A of phospholipid and standards was applied to the PVA-Sil in the phospholipid be well from one The of compounds first from the column, the lipids of The first class of lipids to are We separation a for as a of the minor of this column, but even the from and that and and at the the the phospholipid which is one and the standards not the species are not from their also the of this for the determination of important lipid the and in from a separation of and as and as and as on the PVA-Sil column with the mass The is to that used in was as the its separation and ionization were found to be to those of of the lipid species as a of not were not in the of acid or with the the species were for of classes in the was for and and only showed a of the the the and the of the and the the of the and The use of or as the of the chromatographic was for of or to the in to for ionization were found to with the of the separation, a of in from a was the to the or the mass spectrometer. This addition be to the of the but should not the separation or the A of lipids and a was applied to the PVA-Sil in species were on the column to a certain acid methyl and cholesteryl were from one and with and free fatty The are only in and not in the in The diacylglycerol, and and its were each well It be that normal column were not for the system to be for at 1 the column of the column between This might be specific for this system or of the molecular species of and standards be achieved on a reversed-phase The characterization by LC-MS be on the using of one of the to The was selected that be in the by the of their with the to the at and be by the of the to a associated with the This to in addition to those in a for the for and a for a of mass for be to the signals from both classes of lipids, characterization of the molecular species be achieved in the by the molecular for for with the of the the on the chromatographic of and of the components be in this of the molecular of the components LC-MS on one of the as described The obtained for a normal LDL lipid was compared with that for samples the of lipids from nanospray MS most a of and cholesteryl and a of the mass the of the for lipids on the PVA-Sil column for the and the lipid the is from the nanospray its information is the the of of the lipids by the and be information was by the for both PVA-Sil by differences be between lipids from human and and those from normal for cholesteryl and The to the and the most species were the in the and clear differences were in the components. to the cholesteryl esters, the lipids contained a of and to its the at is from a cholesteryl ester to the the be to the the of for the LDL are in the nanospray MS of the isolated components are the is not are here also a with a of acid and acid be for the Analysis of on the reversed-phase column, using the at high of the fatty as in The a of fatty in the from they are centered on the species are those from normal The and species not of are by of the by this but most be from the found or by the the approach is to the column to a mass capable of the of of signals from with the be components only on the PVA-Sil column and are to be of by the total in the for lipids on the PVA-Sil column and from nanospray MS of the isolated on the mass from and The that the total a good of is found in the of the from and on the dC18 reversed-phase are the of the and the obtained by the to those in the of the in as demonstrated in the the of the is further the to for of the lipids and are to were nanospray MS of the and even in the isolated their were the signals the from to from the lipid on the PVA-Sil column provided additional The molecular of one signals at and to and their also those of the The of species at and are each that they are the the nanospray used for from the of the the only one of the free the of the most species, are to for and for Both that are or Murphy R.C. tandem mass spectrometric of and molecular species of in human Chem. PubMed Scopus Google Scholar). clear differences between the species in and were mixtures of lipid class and were on the PVA-Sil column and the biological and a was the was from to of the here were based on either or for signals at the on the of standards on the of the is for individual lipids and to for lipids, the for the are the molecular species for normal LDL lipids were total and cholesteryl for acylglycerols for and total for of total lipids. lipids were and consisted of total cholesterol, total and total we quantitation using a species, but this lipid This the complex and the of with compounds of the The characterization of lipid samples be with X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (918) Google Scholar, M. Murphy R.C. Electrospray mass spectrometry of phospholipids.Mass Spectrom. Rev. 2003; 22: 332-364Crossref PubMed Scopus (733) Google Scholar). samples are that have or are complex to be with nanospray and LC-MS methods have been for a variety of but a robust LC-MS methodology to characterize a of lipids has been in X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (918) Google Scholar) but the LC-MS methodology here has the that it is based on simple instrumentation that should be in MS The of was to an and methodology for use in it should be easily reproducible and should a for further and instrumentation. 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The solvents are compatible with those used for analysis, that system is for the analysis of lipids in a usually for by here for and the mass obtained the reversed-phase LC-MS experiments using high in the information the molecular species in a to in to the or and to in which are from a high in the also to in the signals as a of in addition and/or methyl The or be applied for better only the of were to be The isolated from the biological samples were also by this nanospray as described (3Bruägger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (734) Google Scholar, 4Han X. Yang J. Cheng H. Ye H. Gross R.W. Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. 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The lipids in this study were obtained from a this not to be characterization of the lipids associated with this species may on the of with certain lipid this one of and lipid from the patient were and the were compared with published for A of samples to be be the were for the lipid in the the molecular each was and the in the lipids was from that of the lipids. each analysis, the total of the normal-phase LC-MS were this allows for a by most lipid in of total differences in the molecular between the and lipids were but they were in the of cholesteryl and The most species in the cases were the they for LDL lipids from lipids. a not the compounds were found to in between and lipids in the of components and were The of minor species or fatty were even in the The only fatty acid species not in in the samples was The at contained the fatty and the of the a of compounds not was their were not to the at The signals of low those at and in the the standards are which are to to of total LDL B. J. as lipid of low PubMed Scopus Google Scholar). species of the total LDL are by MS J. M. M. J. Lipid analysis of human and LDL by mass spectrometry and Lipid 2001; PubMed Google Scholar) or nanospray MS of the samples as a of by in the isolated their are signals from as with the that be to of compounds were at that compounds were to the this to have the experiments compounds to be or are to be of LDL as well as LDL B. J. as lipid of low PubMed Scopus Google Scholar) and are in at (1Murphy R.C. Fiedler J. Hevko J. Analysis of nonvolatile lipids by mass spectrometry.Chem. Rev. 2001; 101: 479-526Crossref PubMed Scopus (235) Google Scholar). approach to structures be acid of the Murphy R.C. tandem mass spectrometric of and molecular species of in human Chem. PubMed Scopus Google this be of a analysis of LDL it that the mutant has a for certain on lipids normal differences are to the or are to be the is to further is generally for complex lipid mixtures. the that ionization in or is the class lipids, the of the the and of of the also a of the has been provided by (3Bruägger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (734) Google used standards to for in a for quantitation of be achieved by Murphy R.C. Analysis of membrane as Lipid 2005; PubMed Scopus (31) Google Scholar). the of is for the and described in this the were only in that the used for the and the characterization is only standards were and for the fatty was to and Gross X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (918) Google in this the is a minor standards were they were not used in the analysis the was not and the species we as a for the might have with compounds to be out with and standards be standards that may be used to quantitative which be used in both on the normal-phase column, or which are in both The quantitation as here to be to its also by the use of instrumentation. an additional we that the of the on the specific by the of the mass were obtained for that not be from one by was with here. The LC-MS methodology a robust and simple method for the investigation of complex lipid mixtures. The method used here for quantitation standards allows for a characterization of the total and be by an choice of The methodology should be easily adaptable for and sensitivity using and MS instrumentation we used for this initial This was by of and and and The are to for
Sommer et al. (Sun,) studied this question.