Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1–2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1–2 nmol/ml, but the response was non-linear.—DeLong, C. J., P. R. S. Baker, M. Samuel, Z. Cui, and M. J. Thomas. Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography. J. Lipid Res. 2001. 42: 1959–1968. Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1–2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1–2 nmol/ml, but the response was non-linear. —DeLong, C. J., P. R. S. Baker, M. Samuel, Z. Cui, and M. J. Thomas. Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography. J. Lipid Res. 2001. 42: 1959–1968. Glycerophospholipids (GPLs) are the basic building blocks for cellular membranes and define cellular and subcellular structures. In addition to being critical components of cellular membranes, GPLs interact with all membrane proteins and many non-membrane proteins as well as mediate signal transduction (1Exton J.H. Phosphatidylcholine breakdown and signal transduction.Biochim. Biophys. Acta. 1994; 1212: 26-42Google Scholar). GPLs contain five structural moieties, including polar head group, phosphoryl group, glycerol backbone, and either a fatty ether or acyl side chains at the sn-1 position and fatty acyl chains at the sn-2 position. The numerous combinations of chain lengths, double bonds, linkages to the glycerol backbone of the side chains, and different head groups enable the formation of an immense number of molecular species. Many of the functional aspects of GPL depend upon these structural subtleties. Given their structural and functional roles in mammalian cells, the understanding of GPL composition, metabolism, and regulation at the level of molecular species has become increasingly important. Conventional strategies for quantitation of GPL molecular species require many steps. The first step is to separate total lipid extracts into lipid subfractions by either TLC or HPLC. Subsequent steps include the removal of the GPL head-group, derivatization of the sn-3 position, and then separation by normal and reverse-phase HPLC. Individual molecular species are collected from the HPLC, then saponified and esterified to produce fatty acid methyl esters and dimethylacetals that are analyzed by gas chromatography (2Warne T.R. Robinson M. A method for the quantitative analysis of molecular species of alkylacylglycerol and diacylglycerol.Lipidsa. 1990; 25: 748-752Google Scholar, 3Blank M.L. Cress E.A. Fitzgerald V. Snyder F. Thin-layer and high-performance liquid chromatographic separation of glycerolipid subclasses as benzoates. Derivatives of ether and ester analogues of phosphatidylcholine, phosphatidylethanolamine and platelet activating factor.J. Chromatogr. 1990; 508: 382-385Google Scholar, 4Blank M.L. Cress E.A. Snyder F. Separation and quantitation of phospholipid subclasses as their diradylglycerobenzoate derivatives by normal-phase high-performance liquid chromatography.J. Chromatogr. 1987; 392: 421-425Google Scholar, 5Blank M.L. Robinson M. Fitzgerald V. Snyder F. Novel quantitative method for determination of molecular species of phospholipids and diglycerides.J. Chromatogr. 1984; 298: 473-482Google Scholar). These prior separations and manipulations are labor-intensive, time-consuming, and may suffer from reduced recovery and selective loss of certain molecular species. Analysis of the unprocessed total lipid extract by electrospray ionization tandem mass spectrometry (ESI-MS/MS) bypasses many of these problems. ESI-MS/MS is a powerful tool for the study of phospholipids because it: 1) requires minute amounts of sample, 2) employs a “soft” ionization procedure that produces mostly singly charged GPL ions (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, 7Kerwin 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, 8Brügger 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 tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar), 3) can process a sample containing a mixture of different GPLs, 4) can distinguish GPL classes and identify individual molecular species via unique collision-induced decomposition pathways (9Kayganich K. Murphy R.C. Molecular species analysis of arachidonate containing glycerophosphocholines by tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 1991; 2: 45-54Google Scholar, 10Cole M.J. Enke C.G. Direct determination of phosphoipid structures in microoranisms by fast atom bombardment triple quadrupole mass spectrometry.Anal. Chem. 1991; 63: 1032-1038Google Scholar, 11Huang Z.H. Gage D.A. Sweeley C.C. Characterization of diacylglycerylphosphocholine molecular secies by FAB-CAD-MS/MS: a general method not sensitive to the nature of the fatty acyl groups.J. Am. Soc. Mass Spectrom. 1992; 3: 71-78Google Scholar, 12Smith P.B.W. Snyder A.P. Harden C.S. Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry.Anal. Chem. 1995; 67: 1824-1830Google Scholar), and 5) is very fast. Thus, ESI-MS/MS methods provide unparalleled speed and precision for the rapid quantitation of GPL mixtures. In this study, we have assessed the effects of TLC and HPLC separation on GPL composition using ESI-MS/MS. This is the first study to provide experimental proof of the changes in molecular species composition that occurs during chromatography. We provide additional detail on sample and instrument parameters that are important for the use of ESI-MS/MS for quantitative analysis of total cellular GPL. Several studies have focused on quantitation of GPL (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, 8Brügger 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 tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar, 13Lehmann W.D. Kessler M. Characterization and quantification of human plasma lipids from crude lipid extracts by field desorption mass spectrometry.Biomed. Mass Spectrom. 1983; 10: 220-226Google Scholar, 14Duffin K. Obukowicz M. Raz A. Shieh J.J. Electrospray/tandem mass spectrometry for quantitative analysis of lipid remodeling in essential fatty acid deficient mice.Anal. Biochem. 2000; 279: 179-188Google Scholar, 15Lehmann W.D. Koester M. Erben G. Kepler D. Characterization and quantification of rat bile phosphatidylcholine by electrospray-tandem mass spectrometry.Anal. Biochem. 1997; 246: 102-110Google Scholar, 16Han X. Gross R.W. Structural determination of picomole amounts of phospholipids via electrospray ionization tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 1995; 6: 1202-1210Google Scholar, 17Waugh R.J. Morrow J.D. Roberts 2nd, L.J. Murphy R.C. Identification and relative quantitation of F2-isoprostane regioisomers formed in vivo in the rat.Free Radical Biol. Med. 1997; 23: 943-954Google Scholar, 18Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria-Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.J. Biol. Chem. 1999; 274: 28395-28404Google Scholar, 19Carrier A. Parent J. Dupuis S. Quantitation and characterization of phospholipids in by liquid spectrometry.J. Chromatogr. A. 2000; Scholar, Electrospray spectrometry separation of as a for ion 2000; Scholar, M. P. R. P. determination of phospholipid by effects of acyl chain and lipid on instrument Lipid Res. 42: Scholar), but these have not the changes in GPL molecular species composition that during chromatography. were from or were from TLC plates were from The HPLC by with was from were from were the from Lipid were extracted from rat liver by the method of and A rapid method of total lipid and J. Biochem. Scholar). phospholipid of the extracts was using the lipid of G. S. Quantitative analysis of phospholipids by chromatography and analysis of Scholar). of total rat liver lipid extract were in at were analyzed by without by TLC and by HPLC. sample was in a of containing chromatography. of lipid extract were by TLC on plates that were in an at for The plates were in a of The from the of TLC was into a and extracted with of by and The extracts were to to and choline (PC) showed the of the was by in of with of acid for of was and the mixture for 1–2 and to The was and with The sample was in a of and in a of containing HPLC. of lipid extract were in and then into phospholipid subclasses by normal HPLC as by The distribution and of phospholipids in cellular J. with a HPLC The was acid in the and at a of GPLs were with a of and The of GPL classes was at The ethanolamine (PE), and PC were the individual components and the in a of The for were then in and at TLC GPLs were extracted from by an and A rapid method of total lipid and J. Biochem. containing The lipid extract was into then by TLC using as the In was in of the plates from the the plates were to for of PC from The was with of by and A rapid method of total lipid and J. Biochem. to Mass spectrometry were by the PC in were with a and TLC plates were in a for The to PC was and extracted as PC was in of of was and the mixture at for additional was and the continued for derivatization by the lipids were extracted by the method of and A rapid method of total lipid and J. Biochem. by ESI-MS/MS of lipid was in containing fatty acid and to in The for at after the lipids were extracted by the method of and A rapid method of total lipid and J. Biochem. Scholar). Electrospray ionization tandem mass GPLs were analyzed on a triple quadrupole mass were using were at a of by a an of and a of for and were and respectively. were at with a of and a of The GPL classes and were analyzed in the ion using from to respectively. species were by the of species were by for a loss of species were by for that a loss of The fatty acid distribution of individual molecular species was in the ion by ion analysis of ions from PC or ions from and Analysis of the charged ions was with from to V. GPL were to nmol/ml in for the of ion has to with ion mass 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 tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar), a mass was by a to a of ion intensity at a GPL The ion intensity was to the first ion in the The experimental ion at were by by the The of ion intensity for PC was linear but a to a The for was the of linear a of and concentrations were Phosphatidylcholine was by the formation of loss of from ions was to PE. of and to signal intensity of PC and PC was more easily of was by including acid in the The PC had a linear response of ion from to nmol/ml the was linear from to nmol/ml these the response was and to liver lipid lipids and was also analyzed for PC and concentrations of total cellular phospholipid in response of PC and molecular species to the with concentrations to and nmol/ml, respectively. Lipid classes are phospholipid molecular species. However, the phospholipid classes are not after chromatography. In the we that the individual molecular species were in to their in the original We this analyzing PC and molecular species by ESI-MS/MS and after chromatographic rat liver lipid extract was by TLC or by normal-phase HPLC. was not phospholipid the ionization of all of the phospholipid were analysis and the phospholipid distribution with the distribution in the extract. were to nmol/ml lipid The loss of phospholipid by chromatography was and for HPLC and respectively. acid to the have the recovery from TLC but the of lipid or shows the contribution of molecular species to the total PC and and and and after TLC and HPLC. The in were for reduced with increase in phospholipid ion shows that was a selective loss of certain phospholipid species after both TLC and HPLC These losses were to the total number of double bonds in the 3 is a of the number of total double bonds in the sn-1 and sn-2 fatty acids the in contribution to total PC or after TLC or HPLC. The of molecular species in double group, or were and the are in The contribution of species 0 to 3 double bonds was reduced after chromatography. The for PC was 20% after TLC separation and 7.2% after HPLC, 19.7% after TLC separation and 7.5% after HPLC. GPLs a total of to double bonds had a greater contribution to the total after the was with that the ion to in the total ion intensity with a in the distribution of PC molecular species in the as with the distribution in the ion The contained ions that had greater than in those in the ion A mass increase of mass of a by the We that are formed by to PC and PC a different distribution of ions as in and C. PC showed a The intensity of the ion from the PC was reduced relative to The of PC was to that by and Gross X. Gross R.W. Structural determination of regioisomers by electrospray ionization tandem mass spectrometry.J. Am. Chem. Soc. Scholar). The loss of in is of the We use the loss to the of in PC X. Gross R.W. Structural determination of regioisomers by electrospray ionization tandem mass spectrometry.J. Am. Chem. Soc. Scholar). can by PC in with of with then the with and Analysis of in this not contain not studies of PC from TLC plates occasionally showed the of molecular mass PCs that were not in the extract. and the ion and after The TLC was to for an additional after to the and for TLC plates were in a to that of lipid extract not to TLC that of the original PC was into molecular mass PCs. A oxidation was This in the loss of a mass of a fatty acid chain and the addition of an the of an the molecular mass PCs were either reduced with or into a with PCs a mass than the the mass of the oxidized was to with PCs greater than the the mass of the oxidized to of unsaturated extract from was oxidized as in and by a TLC to The PC was extracted from and analyzed by mass PC were by ion analysis for A shows a PC from PCs that were not by is the molecular species in this shows the PC ion from PCs that were on a TLC for after A with is the molecular species in this shows the PC after the in with to The of the alcohols are than their to shows the PC generated by the from with The formation of a increase the mass by to The are of separate the composition of oxidized we ion analysis of PCs. This procedure ion of the generated from the (9Kayganich K. Murphy R.C. Molecular species analysis of arachidonate containing glycerophosphocholines by tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 1991; 2: 45-54Google Scholar, 11Huang Z.H. Gage D.A. Sweeley C.C. Characterization of diacylglycerylphosphocholine molecular secies by FAB-CAD-MS/MS: a general method not sensitive to the nature of the fatty acyl groups.J. Am. Soc. Mass Spectrom. 1992; 3: 71-78Google Scholar). The ion analysis of the ion to the ion an at sn-2 and at low molecular mass PCs had ion of and to the ion in the ion of and the of sample GPL on a TLC after the has phospholipid were in on a separate TLC and then has that the of a TLC band may from the of a We ESI-MS/MS to sample of phospholipid classes were or separation of molecular species had different TLC plates were with PC and the plates of the plates was with to PC and the of the TLC band was into These were into separate and the GPL analyzed by ESI-MS/MS. the PC of the PC containing and fatty acids migrated at the front of the PC band in 3 contained chain PCs of a that gives the ion the of PC with molecular mass 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 tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar, M.J. Z. Molecular of phosphatidylcholine between the and phosphatidylethanolamine Biol. Chem. 1999; 274: Scholar). However, by a of ion can mass The effect of on was by analyzing at from to of PC and and and and of phospholipid in mass by The are in as the intensity of the molecular mass by the intensity of the low molecular mass phospholipid the with as in to intensity of and low mass ions a of the total ion intensity not The of this study important and GPL chromatography. at concentrations of GPL than nmol/ml was a linear between ion was a selective loss of GPL molecular species with than 3 double bonds from by either TLC or HPLC. The losses from to 20% and were smaller for HPLC and to greater for oxidation of unsaturated GPL on TLC plates can after to molecular species a GPL on TLC We have also that analysis by ESI-MS/MS bypasses these sample and are critical in of PC and a linear response from to nmol/ml and to nmol/ml respectively. these the response was and to concentrations of total phospholipid from rat liver extracts response of PC and molecular species to the with concentrations at to nmol/ml of total M. P. R. P. determination of phospholipid by effects of acyl chain and lipid on instrument Lipid Res. 42: ion for phospholipid molecular species. showed that the ion to above nmol/ml total of the response above to nmol/ml total we quantitative studies at or nmol/ml than individual for A unique of ESI-MS/MS is the rapid analysis of total lipid extracts without preliminary or The analysis the molecular species composition of different phospholipid subclasses with to were GPL were by chromatography. is that these losses are distributed the phospholipid molecular species. Using we have that the chromatographic to separate phospholipid classes the distribution of molecular species. 3 shows that the distribution of losses phospholipid molecular species was for the more those from 0 to 3 double bonds in the fatty acids. losses to molecular species 0 to 3 double bonds caused an in molecular species from to double molecular more unsaturated PC molecular species migrated in a more than PCs. We that the PCs containing more unsaturated fatty acids have a with the are more in the The by the unsaturated PCs that to to the are a of the GPL that are sensitive to In the of the acid and to into the electrospray are to The not in the mass with the more and PCs and the PCs to on the plates for an of caused the oxidation of unsaturated PCs and the formation of molecular mass PCs. These oxidation reduced to alcohols by suggesting that a was in the formation of that the is an than a of the double bonds to either aldehydes or but not Analysis of of the oxidized PCs showed that contained acids in addition to a fatty that oxidation an the ion may acid and the ion The of ion is with oxidation of the double to the of acid as by and Murphy Murphy R.C. Structural characterization of oxidized phospholipid from Lipid Res. 2000; Scholar). is formed by the oxidation of of a number of PC showed that of the contained amounts of the smaller PCs. of the PCs were in These that the methods for TLC separation amounts of these oxidation However, these amounts may important for studies that in vivo lipid There are of the oxidation The first is that oxidation of PC on TLC plates a of the total mass of unsaturated fatty acids is Analysis by lipid not changes in oxidation that on lipids to in oxidation a However, the GPL are in with the TLC for the become more A more consequence of oxidation during chromatography the formation of minute amounts of oxidized PC those that are to have biological G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria-Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.J. Biol. Chem. 1999; 274: 28395-28404Google Scholar, G.A. Prescott S.M. McIntyre T.M. fragmented phospholipids as the side of 1995; Scholar). The loss of lipid and in molecular species composition caused by sample and chromatographic separation were by analyzing a total cellular lipid extract. However, we that total phospholipid than the of the linear for the of PC quantification in a total cellular in PC of was than that of a PC at or the quantitative between The fatty acyl for were in the ion using collision-induced to The of the ions provide because the intensity is greater than P.B.W. Snyder A.P. Harden C.S. Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry.Anal. Chem. 1995; 67: 1824-1830Google Scholar, Gross M.L. atom bombardment and tandem mass spectrometry of and Scholar). However, to this general and the on G. of in the collision-induced of from glycerophospholipids using ion electrospray tandem quadrupole mass Mass Spectrom. Scholar). was to have a than at a of G. of in the collision-induced of from glycerophospholipids using ion electrospray tandem quadrupole mass Mass Spectrom. Scholar), the phosphatidylcholine had an greater than not as by G. of in the collision-induced of from glycerophospholipids using ion electrospray tandem quadrupole mass Mass Spectrom. Scholar), for GPL with additional of in the ion from the mass of certain and PC molecular species. In the ion PC species a and a methyl from the This loss of can in a molecular mass PC species a ion that is to the ion mass of a molecular mass species. ion analysis in this or four fatty acid is to the fatty acid composition of phospholipid species on the from ion because the of PC and by mass of molecular species composition can by GPL subclasses using HPLC or by a of the A. J. of of lipids their by electrospray ionization tandem mass spectrometry.J. Am. Soc. Mass Spectrom. Scholar). These studies that chromatographic separation of PC and molecular species analysis to losses of certain molecular species. by 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 tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar), analysis of lipid extracts gives an composition for PC and PE. However, for analysis in this the total of species into the electrospray not 1–2 We S. and A. for use of the HPLC and and and C. for rat and were by the and from the of This was in by from the and and from the of The mass was in with from and with from the and of the for from to the of electrospray ionization tandem mass spectrometry glycerophospholipid mass spectrometry triple quadrupole mass spectrometry first quadrupole of a triple quadrupole choline ethanolamine