Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics

Abstract

Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantitate the hundreds of chemically distinct lipids in cells and determine the molecular mechanisms through which they facilitate cellular function. Recent developments in electrospray ionization mass spectrometry (ESI/MS) have made possible, for the first time, the precise identification and quantification of alterations in a cell's lipidome after cellular perturbations. This review provides an overview of the essential role of ESI/MS in lipidomics, presents a broad strategy applicable for the generation of lipidomes directly from cellular extracts of biological samples by ESI/MS, and summarizes salient examples of strategies utilized to conquer the lipidome in physiologic signaling as well as pathophysiologically relevant disease states. Because of its unparalleled sensitivity, specificity, and efficiency, ESI/MS has provided a critical bridge to generate highly accurate data that fingerprint cellular lipidomes to facilitate insight into the functional role of subcellular membrane compartments and microdomains in mammalian cells.We believe that ESI/MS-facilitated lipidomics has now opened a critical door that will greatly increase our understanding of human disease. Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantitate the hundreds of chemically distinct lipids in cells and determine the molecular mechanisms through which they facilitate cellular function. Recent developments in electrospray ionization mass spectrometry (ESI/MS) have made possible, for the first time, the precise identification and quantification of alterations in a cell's lipidome after cellular perturbations. This review provides an overview of the essential role of ESI/MS in lipidomics, presents a broad strategy applicable for the generation of lipidomes directly from cellular extracts of biological samples by ESI/MS, and summarizes salient examples of strategies utilized to conquer the lipidome in physiologic signaling as well as pathophysiologically relevant disease states. Because of its unparalleled sensitivity, specificity, and efficiency, ESI/MS has provided a critical bridge to generate highly accurate data that fingerprint cellular lipidomes to facilitate insight into the functional role of subcellular membrane compartments and microdomains in mammalian cells. We believe that ESI/MS-facilitated lipidomics has now opened a critical door that will greatly increase our understanding of human disease. Lipidomics is a rapidly expanding research field fueled by recent advances in, and novel applications of, electrospray ionization mass spectrometry (ESI/MS). Lipidomics is focused on identifying alterations in lipid metabolism and lipid-mediated signaling processes that regulate cellular homeostasis during health and disease. Research in lipidomics incorporates multiple techniques to quantify the precise chemical constituents in a cell's lipidome, identify their cellular organization (subcellular membrane compartments and domains), delineate the biochemical mechanisms through which lipids interact with each other and with crucial membrane-associated proteins, determine lipid-lipid and lipid-protein conformational space and dynamics, and quantify alterations in lipid constituents after cellular perturbations. Through the detailed quantification of a cell's lipidome (e.g., lipid classes, subclasses, and individual molecular species), the kinetics of lipid metabolism, and the interactions of lipids with cellular proteins, lipidomics has already provided new insights into health and disease. The true power and promise of lipidomics, however, is only beginning to be realized. Decades of painstaking research in the 1970s and 1980s developed a straight and reversed-phase HPLC system that could “rapidly” (30 min) separate phospholipid classes, molecular species, and regioisomers into individual chemical constituents (1Cooper M.J. Anders M.W. High pressure liquid chromatography of fatty acids and lipids.J. Chromatogr. Sci. 1975; 13: 407-411Google Scholar, 2Gross R.W. Sobel B.E. Isocratic high-performance liquid chromatography separation of phosphoglycerides and lysophosphoglycerides.J. Chromatogr. 1982; 197: 79-85Google Scholar, 3Creer M.H. Gross R.W. Separation of isomeric lysophospholipids by reverse phase HPLC.Lipids. 1985; 20: 922-928Google Scholar, 4Creer M.H. Gross R.W. Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids.J. Chromatogr. 1985; 338: 61-69Google Scholar, 5McCluer R.H. Ullman M.D. Jungalwala F.B. HPLC of glycosphingolipids and phospholipids.Adv. Chromatogr. 1986; 25: 309-353Google Scholar, 6Robins S.J. Patton G.M. Separation of phospholipid molecular species by high performance liquid chromatography: potentials for use in metabolic studies.J. Lipid Res. 1986; 27: 131-139Google Scholar). These techniques, however, were labor intensive and required sensitive separation methods for which quantitation was difficult, because the π → π* transition during UV detection was not strictly proportional to mass content (7Gross R.W. High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization.Biochemistry. 1984; 23: 158-165Google Scholar). Moreover, these procedures could not meet the needs for the application of lipidomics to the study of human disease because they were plagued by cumulative errors from multistep chromatographic procedures. Finally, these early efforts required a substantial amount of mass for adequate signal-to-noise ratios and suffered from an inability to accurately quantify minor mass constituents, which are of substantial importance in lipid-mediated signaling processes. These facts notwithstanding, substantial insights were made through HPLC, fast atom bombardment/MS, and gas chromatography/MS approaches in the 1980s (7Gross R.W. High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization.Biochemistry. 1984; 23: 158-165Google Scholar, 8Aberth W. Straub K.M. Burlingame A.L. Secondary ion mass spectrometry with cesium ion primary beam and liquid target matrix for analysis of bioorganic compounds.Anal. Chem. 1982; 54: 2029-2034Google Scholar, 9Lehmann W.D. Kessler M. Fatty acid profiling of phospholipids by field-desorption and fast-atom-bombardment mass spectrometry.Chem. Phys. Lipids. 1983; 32: 123-135Google Scholar, 10May H.E. Desiderio D.M. Fast-atom-bombardment mass spectrometry of underivatized phosphatidylcholines, lysophosphatidylcholines, and diglycerides.J. Chem. Soc. Chem. Commun. 1983; : 72-73Google Scholar, 11Matsubara T. Hayashi A. FAB/mass spectrometry of lipids.Prog. Lipid Res. 1991; 30: 301-322Google Scholar), and the stage was set for the growth of lipidomics in the 1990s. Because the extraordinary sensitivity of ESI/MS for lipid analyses was first identified in the initial analyses of platelet activating factor by Weintraub et al. (12Weintraub S.T. Pinckard R.N. Hail M. Electrospray ionization for analysis of platelet-activating factor.Rapid Commun. Mass Spectrom. 1991; 5: 309-311Google Scholar) and diacylglycerols by Duffin et al. (13Duffin K.L. Henion J.D. Shieh J.J. Electrospray and tandem mass spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar solvents.Anal. Chem. 1991; 63: 1781-1788Google Scholar), multiple ESI/MS techniques have been developed and extensively used for the analyses of various classes, subclasses, and individual molecular species of lipids from biological sources. An excellent and extensive review covering these developments and applications has recently been published (14Murphy R.C. Fiedler J. Hevko J. Analysis of nonvolatile lipids by mass spectrometry.Chem. Rev. 2001; 101: 479-526Google Scholar). Herein, we will provide an overview of the essential role of ESI/MS in the development of lipidomics and present a strategy we have utilized to obviate chromatographic separation of lipids resulting in the unparalleled precision, accuracy, and speed in the determination of individual cellular lipidomes. ESI is an ionization technique used for the mass spectrometric analysis of polar compounds that was initially developed by Fenn and colleagues (15Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Google Scholar). The technique has been extensively explored, and many theories of the physical processes involved in ion production have been proposed and validated in some detail (16Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization—principles and practice.Mass Spectrom. Rev. 1990; 9: 37-70Google Scholar, 17Smith R.D. Loo J.A. Edmonds C.G. Barinaga C.J. Udseth H.R. New developments in biochemical mass spectrometry: electrospray ionization.Anal. Chem. 1990; 62: 882-899Google Scholar, 18Cole R.B. Electrospray Ionization Mass Spectrometry. Wiley, New York1997Google Scholar, 19J. F. de la Mora van Berkel G.J. Enke C.G. Cole R.B. Martinez-Sanchez M. Fenn J.B. Electrochemical processes in electrospray ionization mass spectrometry.J. Mass Spectrom. 2000; 35: 939-952Google Scholar). ESI is effected by applying a strong electric field (∼106 V/m with a potential difference of 3–6 kV) under atmospheric pressure to a liquid passing through a capillary tube with a slow flow (normally 1–10 μl/min). The field induces charge accumulation at the liquid surface located at the end of the capillary, and mechanical forces are utilized to spray the mobile phase into highly charged droplets. A sheath gas flowing coaxially to the infused spray forces dispersion to be limited in space and minimizes the effects of mobile phase differences during online ESI/MS. These droplets then pass either through a curtain of heated inert gases (most often nitrogen) or through a heated capillary, or both, for subsequent desolvation, leaving the generated ions ready for MS analysis. The evaporation of the solvent in droplets causes them to shrink to the point where the repulsive Coulombic forces approach the magnitude of the forces of surface tension, as predicted from the formula of Lord Rayleigh over 100 years ago: (Eq. 1)q2 = 8π2ε0γD3 where q is the of the is the surface tension, and is the of a the Rayleigh is the droplets a of droplets. These droplets then a of and droplets. the solvent is from the the has been R.D. Loo J.A. Edmonds C.G. Barinaga C.J. Udseth H.R. New developments in biochemical mass spectrometry: electrospray ionization.Anal. Chem. 1990; 62: 882-899Google Scholar). ESI be used for that not through of as in as as a potential is present in a to interact with either a or be during the ESI are fatty acids are nonpolar be and with a sensitivity in the through of from ions with the in the infused Gross R.W. in lipid molecular species in J. 2000; Scholar, Gross R.W. analysis and molecular species of molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass 2001; Scholar). The of ESI ionization is on the of each lipid to either or under the high This was to the of lipid directly from extracts chromatographic separation Gross R.W. Electrospray ionization mass spectroscopic analysis of human membrane Sci. Scholar, Gross R.W. in individual molecular species of human platelet phospholipids during electrospray ionization mass identification of the for the magnitude and of platelet phospholipid 35: Scholar). lipid be through their electric multiple chromatographic procedures. Through use of each of lipid be in the ionization and individual molecular species be by MS tandem the in our and of our colleagues have that ESI/MS of lipids of the and methods to alterations in the cellular lipidome Gross R.W. analysis and molecular species of molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass 2001; Scholar, Gross R.W. Electrospray ionization mass spectroscopic analysis of human membrane Sci. Scholar, Gross R.W. in individual molecular species of human platelet phospholipids during electrospray ionization mass identification of the for the magnitude and of platelet phospholipid 35: Scholar, of molecular species of and electrospray mass spectrometry.J. Lipid Res. 35: Scholar, W.D. analysis of biological membrane lipids at the by ionization tandem mass Sci. Scholar, A. J. of of lipids their identification by electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. 9: Scholar, analysis of phospholipids in membrane from cells tandem mass Scholar, and quantitation of molecular species from lipid extracts of biological samples by electrospray ionization tandem mass Scholar, Gross R.W. Lipid are in arachidonic acid and and their is of a electrospray spectrometric Scholar, D.M. Electrospray ionization mass spectrometry analysis of in phospholipids in cells during Sci. 2001; Scholar). Through ESI/MS the analysis of lipid classes, subclasses, and individual molecular species in chromatographic separation or a signal-to-noise in to other mass spectrometric a the of molecular ions and the mass of individual lipids over a of ion on the of the polar lipid or the individual molecular and excellent of of Through of these techniques, a high for the detailed study of lipid alterations has been developed at a disease are in (e.g., and We have that approach is only at of lipid in the in which lipid-lipid interactions and ion are The we have utilized from to of lipid as we have Gross R.W. Electrospray ionization mass spectroscopic analysis of human membrane Sci. Scholar). The these has recently been M. determination of phospholipid by effects of and lipid on Lipid Res. 2001; Scholar). the of lipids in the to the point where lipid-lipid interactions the effects of and on lipid quantitation and be M. determination of phospholipid by effects of and lipid on Lipid Res. 2001; Scholar, A. A. of phospholipid mixtures by electrospray mass ion to Scholar). This is to lipid in the droplets during ionization under these high lipid is highly on the physical of lipids Gross R.W. and membrane distinct conformational 1990; Scholar, Gross R.W. on the molecular of and 1991; Scholar). A used strategy for lipidome analyses from biological samples the for chromatographic separation of lipidomes is in and in the extracts of biological samples be by and by of the individual ion with an (e.g., for phospholipids or for after for effects to the as Gross R.W. in individual molecular species of human platelet phospholipids during electrospray ionization mass identification of the for the magnitude and of platelet phospholipid 35: Scholar, D.M. role for in the of Chem. Scholar) for for has been that molecular species of phospholipids have ionization after for effects for molecular species with to and of Gross R.W. Electrospray ionization mass spectroscopic analysis of human membrane Sci. Scholar). A ESI/MS mass of a myocardial lipid multiple phospholipid molecular species that have been identified by tandem MS W.D. analysis of biological membrane lipids at the by ionization tandem mass Sci. Scholar, Gross R.W. determination of of phospholipids electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. Scholar). be that ESI/MS was utilized of ESI tandem MS for quantitation of lipidome because the technique in for individual molecular species constituents that are highly sensitive to the Gross R.W. analysis and molecular species of molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass 2001; Scholar, W.D. analysis of biological membrane lipids at the by ionization tandem mass Sci. Scholar, and quantitation of molecular species from lipid extracts of biological samples by electrospray ionization tandem mass Scholar, Analysis of and by tandem mass 2000; Scholar). many are of molecular species, tandem mass spectrometry be to the of molecular species as we Gross R.W. determination of of phospholipids electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. ionization mass of a myocardial lipid a ESI mass in the of in the lipid and and ESI mass of the lipid after of myocardial lipids were by a and The of molecular species have been by ESI tandem mass spectrometry to the analyses of and in the lipid in of is to each individual cellular of biological samples to ions for and and to molecular species into the separation of lipid in the electrospray ion molecular species then be directly by with an (e.g., after for effects to the by ESI/MS in A ESI/MS mass of the myocardial lipid used for the of after of a amount of multiple molecular species of ion be ion analysis as W.D. analysis of biological membrane lipids at the by ionization tandem mass Sci. Scholar). of each individual molecular species be identified either by ion ESI tandem MS as Gross R.W. determination of of phospholipids electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. Scholar) or by an technique by ESI tandem mass of potential ions in the ion A ion fingerprint constituents of molecular species of a myocardial lipid used for the of of ion to multiple individual molecular ions be ion ESI tandem mass spectrometric analyses as Gross R.W. determination of of phospholipids electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. Scholar). molecular species be from phospholipid molecular species by lipid extracts with to mass spectrometric analyses as Gross R.W. The primary of myocardial is the of the of Chem. Scholar). and molecular species in the extracts be directly as their by with an after for effects to the in the molecular species in the extracts be directly as their by with an (e.g., after for effects to the in the molecular species be identified by tandem MS W.D. analysis of biological membrane lipids at the by ionization tandem mass Sci. Scholar, A. J. of of lipids their identification by electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. 9: Scholar, Gross R.W. determination of of phospholipids electrospray ionization tandem mass spectrometry.J. Soc. Mass Spectrom. Scholar). A ESI/MS mass of a myocardial lipid used for the of multiple phospholipid molecular species Because of the of some lipid a of in lipid extracts of biological either or separate ESI/MS analyses to be after the lipid by of a amount of in fatty acids in will be to their and be by ESI/MS in through the mass from to quantification is an with or as only a minor in the lipid extracts (e.g., be to is from A ESI mass of a myocardial lipid in the of a amount of used for the of and molecular species has been that and of molecular species distinct in ion ESI tandem mass and quantitation of molecular species from lipid extracts of biological samples by electrospray ionization tandem mass Scholar). A ion to the of mass or is present in the of or molecular species, of mass and during of lipid extracts in the of be to identify and molecular species in the lipid extracts and quantitation of molecular species from lipid extracts of biological samples by electrospray ionization tandem mass Scholar). a ion with to the of mass for of is present in ion mass of with an quantitation of molecular species from extracts of biological samples in with an after for effects be by of mass and quantitation of molecular species from lipid extracts of biological samples by electrospray ionization tandem mass Scholar). ESI tandem MS with of mass of a myocardial lipid in the of a amount of used for the of and over molecular species which be in to an of nonpolar ions in the ESI mass of lipid extracts under the ionization quantitation as their by ESI/MS is however, by the of from and the of multiple molecular species in the of ion we have recently the of from in with of individual fatty acids of to directly quantitate from biological as in detail Gross R.W. analysis and molecular species of molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass 2001; Scholar). of and in the ESI mass of lipid extracts by fatty analyses is by resulting in a detailed molecular species fingerprint of individual molecular species directly from extracts of biological A fingerprint of molecular species of a myocardial lipid in the lipid constituents the importance of the of from and its on leaving the molecular species for analyses of fatty has been well the from of to ion to molecular species the in the are from ion in the MS This as as of each molecular species from lipid extracts and is over a Gross R.W. analysis and molecular species of molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass 2001; Scholar). and quantitation of individual molecular species directly from extracts of biological samples be with an of which has been in our Gross R.W. analysis and molecular species of molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass 2001; Scholar, J.J. M.J. M.J. A. Gross R.W. The by that by Scholar, M. F. A. Gross R.W. A critical role for in the of of by Sci. Scholar, accumulation fatty Sci. Scholar). each of other nonpolar lipids to be and its be by ESI/MS after as W.D. of at the by ionization tandem mass spectrometry.J. Lipid Res. Scholar). other ionization techniques of as atmospheric pressure chemical which has recently been pressure chemical ionization mass spectrometry for analysis of 2001; Scholar), be to these nonpolar The first lipidome analysis directly from lipid extracts of biological samples was over years Gross R.W. in individual molecular species of human platelet phospholipids during electrospray ionization mass identification of the for the magnitude and of platelet phospholipid 35: Scholar). that the of arachidonic acid in human during were and the individual molecular species from which was were Through quantitation of and in the amount of mass and the mechanisms for the were that was that molecular species were present in human for of species, of species, of and of were highly in the of species or of Moreover, of these species for of species and of the phospholipid of a of of was This the of from only from and of mass of the mass from the was from the of These set a of for the of in human of individual molecular species and of the during platelet This study the importance of as the of in and as the of after of human first application of ESI/MS to lipidome ESI/MS were with HPLC methods R.W. Sobel B.E. Isocratic high-performance liquid chromatography separation of phosphoglycerides and lysophosphoglycerides.J. Chromatogr. 1982; 197: 79-85Google Scholar, 3Creer M.H. Gross R.W. Separation of isomeric lysophospholipids by reverse phase HPLC.Lipids. 1985; 20: 922-928Google Scholar, 4Creer M.H. Gross R.W. Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids.J. Chromatogr. 1985; 338: 61-69Google Scholar). of ESI/MS over HPLC were from these early individual molecular species of phospholipid as and could be in Gross R.W. in individual molecular species of human platelet phospholipids during electrospray ionization mass identification of the for the magnitude and of platelet phospholipid 35: by ESI/MS, the limited sensitivity present with HPLC approaches made the alterations in and in human by by ESI/MS and HPLC were ESI/MS could many individual molecular species HPLC could its to lipid and in Gross R.W. in individual molecular species of human platelet phospholipids during electrospray ionization mass identification of the for the magnitude and of platelet phospholipid 35: in myocardial lipid metabolism have been with during the ESI/MS techniques were to a detailed analysis of the lipidome in Gross R.W. in lipid molecular species in J. 2000; Scholar). The of to alterations in lipid constituents in was and the mechanisms through which they are were identified Gross R.W. in lipid molecular species in J. 2000; Scholar). alterations in lipids from by of were a of molecular species in an over increase in mass and a in molecular species fatty the myocardial mass in in to was a in myocardial content in to Finally, a in in the of a in or myocardial the of each of the alterations in phospholipid classes, subclasses, and individual molecular the alterations in molecular species were not by after of These alterations in myocardial lipid metabolism in the into that either be by or are to by the study of lipidomics has already to new insights into the and the importance potential of fatty acids and their metabolic Through the detailed study of the lipidome, new insights into and the effects of and in cellular metabolism and were first alterations in the lipidome of with in disease were by ESI/MS techniques D.M. in early disease and in molecular characterization electrospray ionization mass spectrometry.J. 2001; Scholar, D.M. J. and in early potential role in disease Scholar). A of alterations in lipidome of and from and of human with of was of lipids at the stage of were a of mass to of in in early in to D.M. in early disease and in molecular characterization electrospray ionization mass spectrometry.J. 2001; Scholar). A of the in mass with the is present of at early stage to of at in of D.M. in early disease and in molecular characterization electrospray ionization mass spectrometry.J. 2001; Scholar). a of the were by and in and in from with D.M. J. and in early potential role in disease Scholar). alterations in the lipidome of to was the of content in the of from early D.M. J. and in early potential role in disease Scholar). alterations in other lipid classes, and were present at early and a of these lipidomes in D.M. in early disease and in molecular characterization electrospray ionization mass spectrometry.J. 2001; Scholar, D.M. J. and in early potential role in disease Scholar). These that alterations in lipidomes an role in the of and be with early in the of and in Because of its unparalleled sensitivity, high and high efficiency, ESI/MS has as a research in lipidomics, our of the role of lipid alterations in disease states. the in ESI/MS techniques, alterations in the lipidome be directly from extracts of biological Through analysis of lipid mass the biochemical mechanisms disease are now beginning to be with high during the our understanding of the biochemical mechanisms disease has been rapidly is now that the power of lipidomics is beginning to be realized. This was by of and The are to the Mass which is by of and for the use of the electrospray ionization mass arachidonic acid disease electrospray ionization fatty acid mass spectrometry