Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response

Abstract

Electrospray ionization-mass spectrometry (ESI-MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecular species are detected with equal efficiency. We studied this and other issues that need to be taken into account to obtain truly quantitative compositional data. The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid species decreased with increasing acyl chain length. This effect became increasingly prominent with increasing overall lipid concentration. Second, the degree of acyl chain unsaturation also had a significant effect on instrument response. At the highest concentration studied (10 pmol/μl), polyunsaturated species gave 40% higher intensity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive dilution. Third, the instrument response for the different head group classes varied markedly depending on the infusion solvent used. Notably, inclusion of ammonia in the infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spectra. The fact that instrument response is dependent on many structural features, overall lipid concentration, solvent composition, and instrument settings makes it necessary to include several internal standards for each phospholipid class to obtain accurate data. Preferably, both unsaturated and saturated standards should be used. Finally, we quantified the major phospholipid classes of BHK cells using ESI-MS. The data agreed closely with those obtained with thin-layer chromatography and phosphorus analysis. This study indicates that quantitative compositional data can be obtained with ESI-MS, provided that proper attention is paid to experimental details, particularly the choice of internal standards. —Koivusalo M., P. Haimi, L. Heikinheimo, R. Kostiainen, and P. Somerharju. Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response. J. Lipid Res. 2001. 42: 663–672. Electrospray ionization-mass spectrometry (ESI-MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecular species are detected with equal efficiency. We studied this and other issues that need to be taken into account to obtain truly quantitative compositional data. The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid species decreased with increasing acyl chain length. This effect became increasingly prominent with increasing overall lipid concentration. Second, the degree of acyl chain unsaturation also had a significant effect on instrument response. At the highest concentration studied (10 pmol/μl), polyunsaturated species gave 40% higher intensity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive dilution. Third, the instrument response for the different head group classes varied markedly depending on the infusion solvent used. Notably, inclusion of ammonia in the infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spectra. The fact that instrument response is dependent on many structural features, overall lipid concentration, solvent composition, and instrument settings makes it necessary to include several internal standards for each phospholipid class to obtain accurate data. Preferably, both unsaturated and saturated standards should be used. Finally, we quantified the major phospholipid classes of BHK cells using ESI-MS. The data agreed closely with those obtained with thin-layer chromatography and phosphorus analysis. This study indicates that quantitative compositional data can be obtained with ESI-MS, provided that proper attention is paid to experimental details, particularly the choice of internal standards. —Koivusalo M., P. Haimi, L. Heikinheimo, R. Kostiainen, and P. Somerharju. Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response. J. Lipid Res. 2001. 42: 663–672. Compositional analysis of phospholipids is of considerable interest because they are involved in many crucial cellular functions such as signal transduction, apoptosis, and protein sorting (1Martin T.F. Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking.Ann. Rev. Cell Dev. Bio. 1998; 14: 231-264Google Scholar, 2Bevers E.M. Comfurius P. Dekkers D.W. Zwaal R.F. Lipid translocation across the plasma membrane of mammalian cells.Biochim. Biophys. Acta. 1999; 1439: 317-330Google Scholar). At present, the analysis is complicated by the low sensitivity and complexity of the commonly available methods. Regarding the former, hundreds of nanomoles of total phospholipid are needed to establish the molecular species profiles of the different classes (3Patton G.M. Fasulo J.M. Robins S.J. Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography.J. Lipid Res. 1982; 23: 190-196Google Scholar). This precludes detailed analyses when subcellular fractions obtained from cultured cells, for instance, are studied. The complexity, on the other hand, derives from the fact that several chromatographic steps and/or enzymatic reactions are required to establish the molecular species patterns. In view of these complications, it is hardly surprising that, so far, a complete molecular species profile (i.e., including all major classes) has been quantitatively determined for very few, if any, mammalian cells or membranes. Mass spectrometry (MS) offers an attractive alternative for the analysis of phospholipid compositions because of its high sensitivity, specificity, and (apparent) simplicity. However, MS has rarely been used for this purpose until very recently. The main reason for this is that the ionization methods previously available (fast-atom bombardment, etc.) cause extensive fragmentation of the lipid molecules; this, along with the extreme complexity (hundreds of different molecular species) of most biological samples, has precluded compositional analysis. The recent introduction of “soft” ionization methods has opened completely new vistas in this field. In particular, electrospray ionization-mass spectrometry (ESI-MS) has been shown to be a very promising technique (4Kerwin J.L. Tuininga A.R. Ericsson L.H. Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.J. Lipid Res. 1994; 35: 1102-1114Google Scholar, 5Kim H.Y. Wang T.C. Ma Y.C. Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization.Anal. Chem. 1994; 66: 3977-3982Google Scholar, 6Han X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar, 7Myher J.J. Kuksis Electrospray MS for lipid Scholar, X. Gross R.W. in individual molecular species of human phospholipids electrospray ionization mass of the for the and of phospholipid 35: Scholar, R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, Ma J. Electrospray ionization mass analyses of phospholipids from and human and subcellular to other and for membrane in 1998; Scholar, R. R. Electrospray ionization mass spectrometry analysis of the lipid molecular species of subcellular acyl of molecular species to the plasma 1999; Scholar, Ma J. Electrospray analyses of human glycerophospholipids and that is with membrane lipid that Chem. Scholar). However, are many issues to be can a tool for quantitative determination of lipid key is that different molecular species are not detected with equal X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar, R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, Quantitative analysis of phospholipids in membrane from cells using mass 1999; Scholar). instance, it has been shown that the sensitivity can markedly on the acyl chain R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). However, other to be studied. of it is not and the instrument response on the degree of unsaturation of phospholipid acyl Second, it is or not the response are by the overall lipid concentration. The data on the effect of the phospholipid head group on instrument response are In this we these and other issues that need to be when using for quantitative determination of and standards were obtained from from and from from species obtained from and high thin-layer chromatography from with a or were from and the as previously R. and of 35: Scholar, of with and a study of the effect of the chain J. 1999; Scholar). The using a of lipid classes in high and using Lipid Res. by on an with in and species were from by obtained from and were from the by using J. P. and of glycerophospholipids in human to Scholar). The were on a as previously high-performance liquid chromatographic for the and of lipid to Sci. Scholar). were of high liquid chromatography or and were from or were in in to to to different were The compositions of these are in the and were from these by or the of the cellular phospholipid a of the internal standards and cells were cultured on in with and lipids were J.M. for the and of total lipids from Chem. in and the into used to the total phospholipid of the E.M. determination of in the and of the used for lipid class analysis by S.J. and of of phospholipid by and J. 1999; and phosphorus analysis E.M. determination of in the and of and the with and internal standards and used for analysis. were both with an ion instrument and a instrument The lipids were in with or or and were to the electrospray a using a the of the ion used as the and the The of the and were to and each were the the and in positive and ion used as the and used as the and The in the MS from to and in from to mass the of ion in the a of The phospholipid species were on the of fragmentation and ion or In the positive ion mode, and the of or and sphingomyelin can be by for of R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, and of by mass Scholar, J.J. Electrospray and mass of that are in Chem. and species can be detected by for of in the ion R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). The instrument response can depending on the phospholipid acyl chain R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). account for this and for the several internal standards were can be by the intensity of the internal standards The concentration of an species can be obtained from the is the concentration of the internal standards and and are the and intensity of the At low total lipid the instrument response is a of thus is of the At high total lipid the a the chain The degree of lipid acyl chain unsaturation can also the instrument response. In it is to for this by including unsaturated internal standards as However, because such standards were not available for all lipid classes the were low total lipid the effect of unsaturation is eliminated or greatly diminished classes were on using as the solvent S.J. and of of phospholipid by and J. 1999; Scholar). The lipid were the and phosphorus determined E.M. determination of in the and of Scholar). study that the acyl chain of can a significant effect on the instrument response R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). study this in an of standards saturated and species and with the ion from the response decreased markedly and in a nearly with increasing acyl chain for both the saturated and Notably, the instrument response for the unsaturated species higher than for the saturated This effect of acyl chain unsaturation be studied were obtained with the and also for and not study the total phospholipid concentration the chain of instrument an of saturated species to or species total At the highest concentration (10 pmol/μl), the response decreased and in a with the acyl chain is in with the study R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). However, when the concentration decreased to total and the response an of the chain length. At the the and chain species the response for higher than for it higher We also studied the chain of instrument response with species to the saturated the response decreased with acyl chain length, and the became prominent with increasing lipid concentration. However, the response a of the acyl chain length, the highest concentration studied (10 data were obtained with and both with the ion and the instrument not study the effect of lipid unsaturation and concentration on the instrument response in an of including species with or can be in the response markedly dependent on lipid unsaturation, particularly the higher instance, species total the response for the polyunsaturated species 40% higher than that for the fully saturated ones. However, when the the effect of unsaturation and disappeared species of total The of the phospholipid head group on instrument response in has been studied but the are In the response for to other phospholipids has not been studied using acyl chain an of and to a degree in with or and were obtained with the ion instrument or positive ion In the ion with as the were major in instrument response as shown in The highest response for and gave and and the adduct of gave low of ammonia in the infusion solvent markedly the response for and to other species Notably, the not with lipid concentration, of ammonia or not and In the positive ion mode, with as the the highest for by the of and other lipids is that in the of and most other the prominent than the the for ammonia the At this the intensity of that of the because these is a and the other lipids as The varied with the total lipid concentration and that, in with of involved in the of from glycerophospholipids using ion electrospray mass Mass 1998; the of instrument can markedly the from those shown in not The of instrument response is an in quantitative analysis. shown that a response can be obtained with a instrument of X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar, and of by mass Scholar). this is the with an ion an of species in chain and unsaturation and using the ion shown in the were dependent on the lipid concentration to but to higher signal is a in and from the of the of the by the the were dependent on the concentration to as shown for species in were obtained for the and not such is also an of species and and to from to with BHK total lipid of total phospholipid and with the ion The response a the concentration studied for each species not a response a considerable of can be obtained for in the of other lipids and other in the lipid determined the effect of structural and lipid concentration on instrument we used this to quantitatively the molecular species profiles of the major phospholipids of this a total lipid and with a of internal standards for each major phospholipid and with the instrument using R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). are shown in unsaturated standards were not available for all phospholipid classes and saturated standards were used to for acyl chain of instrument but not for the unsaturation this the were until the not This as by the need to for unsaturation The molecular species compositions of the major classes are shown in The major species were and the major species were and The most species were and the most prominent species were and significant were those with of and to lipids with a and a or However, of the should also of as these been to be in BHK cells J. of subcellular from cultured BHK cells.Biochim. Biophys. Acta. Scholar). that the MS used (i.e., head or ion not the of the molecular species the of individual species of major classes in of a of a species of the total in a phospholipid an of varied from species) to on lipid to a species with an or an chain in the of the on on on on on on on on on on on on on molecular species were and quantified as detailed in and The of the major species are shown in of a species of the total in a phospholipid an of varied from species) to lipid to a species with an or an chain in the of the Identification on in a new The molecular species were and quantified as detailed in and The of the major species are shown in the of the MS a lipid of BHK cells and into by the using internal standards and class ion and The to phospholipid class analysis by the used for the determination of the total lipid phosphorus shown in the of most phospholipid classes as determined by the MS very with those determined by the in this study or previously J. of subcellular from cultured BHK cells.Biochim. Biophys. Acta. Scholar). This is particularly for the and The by the MS from the fact that the and in than the species when determined using the of mass in the ion positive R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, Ma J. Electrospray analyses of human glycerophospholipids and that is with membrane lipid that Chem. Scholar). We to the species by for of in the ion mode, because it has been that this is species R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar). However, for not be The of standards precluded of this In of the MS a higher than the This be to the fact that extensive of the of the effect not This in of the of the cells, as the saturated internal standards than the of the of the lipid BHK cells of total the data shown in it can be that the MS a of for the of and the and not be in the analysis to of of total BHK phospholipids J. of subcellular from cultured BHK cells.Biochim. Biophys. Acta. the MS for the total phospholipid This is to the by the determination and thus the of the MS data. This study that in ESI-MS, the instrument response for phospholipids can on many include acyl chain length, acyl chain unsaturation, of the head total lipid concentration, solvent composition, and instrument of also been by be The acyl chain to a effect on instrument the lipids detected with higher sensitivity than the this has been previously R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. the study to be the to that the effect is markedly dependent on lipid concentration, it became with increasing lipid concentration. In the of the instrument response acyl chain not but from to when lipid concentration The chain of the instrument response higher lipid to the ionization than ion or other the mass and/or the on the other hand, are to be for the of response with increasing acyl chain We that higher lipid the of the and such the with acyl in the and are than the with a acyl The alternative that the species to of the species in the infusion solvent very for the all used were completely is to be because are commonly to be in and the of the species with the saturated in and to be than the highest concentration of this used by of The most of the study is that unsaturation of phospholipid acyl can a major effect on instrument response this is not that a effect has been previously with using electrospray J.J. Electrospray and mass of that are in Chem. or ionization P. P. of by high-performance liquid ionization mass it not to been in MS on of reason for this is that the effect of acyl chain unsaturation is concentration dependent and has when with used low The of the unsaturation effect into account is by the that the response can by as as 40% depending on the total of and the lipid concentration the is or progressive of the the of the of the molecular as we with biological the unsaturation effect not be fully eliminated by dilution. is not fully the instrument response is by acyl chain is that the unsaturated species the saturated and thus the the in the thus the of the to This be to the of unsaturated lipid from to the phospholipid of a quantitative Scholar). The of the head group also to a major effect on the instrument response In the ion mode, the phospholipids and gave a higher response than or This with a study Quantitative analysis of phospholipids in membrane from cells using mass 1999; is not because the lipids are of ammonia in the infusion solvent markedly the response for because the higher of the group of X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar). sodium or has been used to this effect X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar, Ma J. Electrospray ionization mass analyses of phospholipids from and human and subcellular to other and for membrane in 1998; Scholar, J. of of lipids by electrospray ionization mass spectrometry.J. Mass 1998; Scholar, Ma J. Electrospray ionization mass analyses of phospholipids from to and effects of on phospholipid and Biophys. Acta. Scholar). In study X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google and were to very and gave a response. not to be in with the but this is to in experimental the of in the infusion different lipid concentration, or different instrument In the and when using as the major to the species were detected along with the This also previously (4Kerwin J.L. Tuininga A.R. Ericsson L.H. Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.J. Lipid Res. 1994; 35: 1102-1114Google Scholar, 5Kim H.Y. Wang T.C. Ma Y.C. Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization.Anal. Chem. 1994; 66: 3977-3982Google Scholar, X. Gross R.W. determination of of phospholipids electrospray ionization mass spectrometry.J. Mass Scholar, J. determination and quantitative analysis of phospholipids using liquid spectrometry.J. 1998; can greatly the analysis of biological it is that inclusion of ammonia in the infusion solvent greatly the formation of the sodium sodium or be to formation of J. of of lipids by electrospray ionization mass spectrometry.J. Mass 1998; Scholar). However, we not been to obtain of the by inclusion of sodium is that sodium not when a ion is used to the lipids R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, of the and Chem. 1999; this not to be the for and for major sodium adduct were However, inclusion of ammonia eliminated the thus greatly simplifying the interpretation of the not The fact that the instrument response is to such a of including lipid structural solvent and instrument makes it to internal standards when truly quantitative data are of internal standards for each head group class to be so that the acyl chain can be standards be used if the can be to very low total lipid However, of the unsaturation effect not be obtained if or other are In extensive of the molecular those of and because these classes a of the total phospholipids in it to as they a response to that of the the most accurate both unsaturated and saturated standards should be as can fully for the chain and of concentration. In this most were with both an ion and a for both were for the higher sensitivity of the ion However, with samples, the instrument has the significant of analysis of lipid by using ion or R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, and of by mass Scholar, of the and Chem. 1999; Scholar, J. of as a adduct by mass spectrometry with electrospray Mass 35: Scholar). a is particularly when the lipids are the ion of lipid is to extensive of species to different head group The of head also the analysis to the MS Quantitative analysis of phospholipids in membrane from cells using mass 1999; Scholar). Finally, such with a and tool for on phospholipid as been of the and Chem. 1999; Scholar). The major of this is that the detailed of the including the of the individual acyl and be the ion and other mass are to or ion are to and ion of the phospholipid classes is necessary with most to is most by on H.Y. Wang T.C. Ma Y.C. Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization.Anal. Chem. 1994; 66: 3977-3982Google Scholar, J. determination and quantitative analysis of phospholipids using liquid spectrometry.J. 1998; Scholar, of the high-performance liquid mass spectrometry for the analysis of phospholipid molecular species in or Ma J. Electrospray ionization mass analyses of phospholipids from and human and subcellular to other and for membrane in 1998; Scholar, Ma J. Electrospray analyses of human glycerophospholipids and that is with membrane lipid that Chem. Scholar, Ma J. Electrospray ionization mass analyses of phospholipids from to and effects of on phospholipid and Biophys. Acta. Scholar). In the should be when the depending on the chromatographic the of the standards can be different from each other as as from those of the lipids of phospholipid class is that the individual molecular species can be determined by using fragmentation analysis (4Kerwin J.L. Tuininga A.R. Ericsson L.H. Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.J. Lipid Res. 1994; 35: 1102-1114Google Scholar, 6Han X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar, R. Quantitative analysis of biological membrane lipids the low by ionization mass Natl. Acad. Sci. USA. in Natl. Acad. Sci. USA. Scholar, Ma J. Electrospray ionization mass analyses of phospholipids from and human and subcellular to other and for membrane in 1998; Scholar, Ma J. Electrospray ionization mass analyses of phospholipids from to and effects of on phospholipid and Biophys. Acta. Scholar). the of the individual acyl can be determined of involved in the of from glycerophospholipids using ion electrospray mass Mass 1998; Scholar, J. of of lipids by electrospray ionization mass spectrometry.J. Mass 1998; Scholar, X. Gross R.W. determination of of phospholipids electrospray ionization mass spectrometry.J. Mass Scholar, J. determination and quantitative analysis of phospholipids using liquid spectrometry.J. 1998; Scholar, J.J. determination of acyl chain in by high liquid mass Mass 1998; Scholar). However, a truly quantitative determination of the individual molecular species in is very because the of of the and can on the acyl chain and unsaturation of the individual acyl of involved in the of from glycerophospholipids using ion electrospray mass Mass 1998; Scholar, J. of of lipids by electrospray ionization mass spectrometry.J. Mass 1998; Scholar, X. Gross R.W. determination of of phospholipids electrospray ionization mass spectrometry.J. Mass Scholar). of classes is also necessary when but phospholipids such as or a key in lipid J. 1999; Scholar, J. in Cell 1999; are to be as to be available for these be necessary when the high of or other with the ionization Quantitative analysis of phospholipids in membrane from cells using mass 1999; Scholar). Finally, it should be that of the data analysis is to the in quantitative analysis of phospholipid compositions, particularly in of many in the MS a for this in the We are to and for most with the to for in of sphingomyelin and to for This study by from the the of and the to