Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography
2001; Elsevier BV; Volume: 42; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)31524-8
ISSN1539-7262
AutoresCynthia J. DeLong, Paul R.S. Baker, Michael P. Samuel, Zheng Cui, Michaël Thomas,
Tópico(s)Lipid metabolism and biosynthesis
ResumoUsing 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 pharmaceutical formulations by liquid chromatography-mass spectrometry.J. Chromatogr. A. 2000; 876: 97-109Google Scholar, 20Lytle C.A. Gan Y.D. White D.C. Electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids: piperidine as a post column modifier for negative ion detection.J. Microbiological Methods. 2000; 41: 227-234Google Scholar, 21Koivusalo M. Haimi P. Heikinheimo L. Kostianinen R. Somerharju P. Ouantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response.J. Lipid Res. 2001; 42: 663-672Google Scholar), but these reports have not addressed the changes in GPL molecular species composition that take place during chromatography. Phospholipid standards were from Avanti Polar Lipids. Solvents (HPLC or Optima grade) were from Fisher Scientific. Silica gel H TLC plates were from Analtech, Inc. The Supelcosil LC-Si HPLC column, 4.6 mm diameter by 250 mm length packed with 5 micron particles, was from Rainin Instrument, Inc. MDA-231 cells were from ATCC. All other reagents were the highest commercial grade available from Fisher Scientific. Lipid extraction. Lipids were extracted from rat liver by the method of Bligh and Dyer (22Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). Total phospholipid content of the extracts was determined using the lipid phosphorus assay of Rouser et al. (23Rouser G. Siakotas A.N. Fleisher S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.Lipids. 1966; 1: 85-86Google Scholar). Phospholipid recovery. Ten aliquots of total rat liver lipid extract were prepared in methylene chloride at 150 nmol phospholipid/sample. Three aliquots were analyzed by MS/MS without manipulation, three separated by TLC before analysis, and three by HPLC. For analysis, each sample was reconstituted in a solution of methylene chloride–methanol–water 45:45:10 (v/v/v) containing 1% formic acid. Thin layer chromatography. Three 150 nmol aliquots of lipid extract were separated by TLC on silica gel H plates that were dried in an oven at 110°C for 2 h. The loaded plates were developed in a solvent system of chloroform–methanol–ammonium hydroxide 65:35:8 (v/v/v). The silica from the entire length of each TLC lane was scraped into a glass tube and extracted twice with 2 ml of chloroform–methanol–water 2.5:2:1 (v/v/v) followed by two Bligh and Dyer extractions. The combined extracts were centrifuged to remove residual silica, transferred to clean glass tubes, and dried under nitrogen. If choline glycerophosphate (PC) samples showed the presence of sodium adducts, the sodium was removed by treating samples dissolved in 100 μl of methylene chloride with 10 μl of formic acid for 5 min. An equal volume of water was added and the mixture vigorously vortexed for 1–2 min and allowed to stand 5 min. The organic layer was removed and diluted 1:2 with methanol plus 5 μl formic acid. The sample was immediately dried in a stream of nitrogen and dissolved in a solution of methylene chloride–methanol–water 45:45:10 (v/v/v) containing 1% formic acid. HPLC. Three 150 nmol aliquots of lipid extract were dried down, resuspended in hexane–isopropanol–water 2:2.7:0.15 (v/v/v), and then separated into phospholipid subclasses by normal phase HPLC as described by Surette et al. (24Surette M.E. Chilton F.H. The distribution and metabolism of arachidonate-containing phospholipids in cellular nuclei.Biochem. J. 1998; 330: 915-921Google Scholar) with a 4.6 mm × 250 mm Supelcosil LC-Si HPLC column. The eluant was 2-propanol–ethanol–phosphate buffer-pH 7.4–hexane–acetic acid in the following compositions: 367:100:30:490:0.2 (v/v/v/v/v) (Solvent A) and 367:100:50:490:0.6 (Solvent B) at a flow rate of 1 ml/min. GPLs were eluted with a gradient of 0% B (5 min), 0–100% B (10 min), and 100% B (75 min). The elution of GPL classes was monitored at 203 nm. The neutral lipid, ethanolamine glycerophosphate (PE), serine glycerophosphate (PS), inositol glycerophosphate, and PC fractions were collected, the individual components pooled, and the solvent removed in a stream of nitrogen. The combined fractions for each run were dried down, then dissolved in methylene chloride methanol and stored at −70°C. TLC separation. GPLs were extracted from ~1.0 × 107 MDA-231 cells by an acidified Bligh and Dyer extraction (22Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) containing 0.7% acetic acid. The lipid extract was divided into several aliquots, then separated by TLC using chloroform–methanol–ammonium hydroxide 65:35:8 (v/v/v) as the mobile phase. In some studies, methylene chloride was used in place of chloroform. After removing the plates from the development tank, the plates were allowed to stand for various lengths of time before scraping PC fractions from each lane. The silica gel was mixed with 1 ml of 1 M NaCl followed by Bligh and Dyer extraction (22Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) to recover lipids. Mass spectrometry (MS) samples were prepared by dissolving the isolated PC in 500 μl methylene chloride–methanol–water 45:45:10 (v/v/v). Residual particulates were removed with a 2 μm stainless steel filter. Derivatives: sodium borohydride reduction and dinitrophenylhydrazone formation. Developed TLC plates were dried in a fume hood for 12 h. The region corresponding to PC was scraped and extracted as described above. Approximately 1 mg PC was dissolved in 400 μl of 95% ethanol, 100 μl of 0.1 M NaBH4 was added, and the mixture incubated at room temperature for 30 min. An additional 100 μl 0.1 M NaBH4 was added and the reaction continued for 30 min. For derivatization by DNPH, the lipids were extracted by the method of Bligh and Dyer (22Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) followed by ESI-MS/MS analysis. Approximately 1 mg of lipid was dissolved in 100 μl Tris-HCl (0.2 M, pH 7.5) containing 2 mg/ml fatty acid free BSA and added to 500 μl 1.8 mM DNPH in 1 N HCl. The reaction proceeded for 2 h at room temperature, after which the lipids were extracted by the method of Bligh and Dyer (22Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). Electrospray ionization tandem mass spectrometry. GPLs were analyzed on a Micromass Quattro II triple quadrupole mass spectrometer. Data were acquired using MassLynx NT software. All analyses were performed at a flow rate of 5 μl/min provided by a Harvard Apparatus model 55-2111 syringe pump, an argon pressure of 1.8 × 10 −3 mBar, and a source temperature of 200°C. Typical values for capillary and cone voltages were 4.5 kV and 85 V, respectively. Data were recorded at 16 points/Da with a scan time of 1.00 s and a scan delay of 0.10 s. The GPL classes PC, PE, and PS were analyzed in the positive ion mode using collision energies ranging from 15 to 40 V, respectively. PC-containing species were discriminated by measuring the precursors of m/z 184. PE-containing species were detected by scanning for a neutral loss of 141 Da. PS-containing species were detected by scanning for molecules that underwent a neutral loss of 185 Da. The fatty acid distribution of individual molecular species was determined in the negative ion mode by product ion (daughter ion) analysis of [M-CH3]5 ions from PC or [M-H]− ions from PE and PS. Analysis of the negatively charged product ions was performed with collision energies ranging from 15 to 40 V. GPL samples were reconstituted to 2 nmol/ml in methylene chloride–methanol–water 45:45:10 (v/v/v) for analysis. Because the sensitivity of ion detection has been shown to vary with ion mass (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), a mass dependent correction was established by fitting a line to a plot of ion intensity at a constant molar GPL concentration versus m/z. The ion intensity was normalized to the first ion in the spectrum. The experimental ion intensities at each m/z were corrected by dividing by the fitted equation. The relationship of ion intensity versus m/z for PC was roughly linear but gave a better fit to a polynomial equation: 10.494 + (−2.20 × 10−2*m/z) + (1.182 × 10−5*m/z2). The relationship for PE was exponential: 0.372 + 8709.8*exp (−0.0131*m/z). To establish the range of linear response, a series of 1,2-O-tetradecanoyl-sn-glycero-3-phosphocholine, di-14:0 PC, and 1,2-O-hexadecanoyl-sn-glycero-3-phosphoethanolamine, di-16:0 PE concentrations were analyzed. Phosphatidylcholine was detected by the formation of m/z 184 ions, while neutral loss of 141 Da from precursor ions was used to measure PE. At equal concentrations, employing collision energies of 25 and 20 eV to optimize signal intensity of PC and PE, respectively, PC was more easily detected. Ionization efficiency of PE was substantially increased by including 1% formic acid in the solvent. The PC standard had a linear response of ion count versus concentration from 0.004 to 2 nmol/ml (Fig. 1A), while the PE standard was linear from 0.1 to 1 nmol/ml (Fig. 1B). Beyond these concentrations, the response was nonlinear and began to plateau. Rat liver lipid extract, which includes neutral lipids and GPL, was also analyzed for PC (Fig. 1C) and PE (Fig. 1D). Increasing concentrations of total cellular phospholipid resulted in response curves of major PC and PE molecular species similar to the standards with optimum concentrations up to 1 and 2 nmol/ml, respectively. Lipid classes are routinely separated before quantifying phospholipid molecular species. However, the phospholipid classes are not quantitatively recovered after chromatography. In the past, we assumed that the individual molecular species were recovered in proportion to their concentration in the original sample. We tested this assumptionby analyzing PC and PE molecular species by ESI-MS/MS before and after chromatographic separation. Total rat liver lipid extract was separated by TLC or by normal-phase HPLC. Because it was not known whether one phospholipid class influences the ionization efficiency of other classes, all of the phospholipid fractions were pooled before MS analysis and the phospholipid distribution compared with the distribution in the untreated extract. All samples were diluted to 2 nmol/ml lipid phosphorus before analysis. The overall loss of phospholipid by chromatography was approximately 30% and 50% for HPLC and TLC, respectively. Adding acid to the extraction solvents would have undoubtedly improved the recovery from TLC plates, but increased the chance of catalyzing lipid hydrolysis or oxidation. Figure 2 shows the percent contribution of each molecular species to the total PC (A and C) and PE (B and D) before and after TLC and HPLC. The percentages shown in Fig. 2 were corrected for reduced transmission efficiency with increase in phospholipid ion mass. Figure 2 shows that there was a selective loss of certain phospholipid species after both TLC and HPLC separation. These losses were proportional to the total number of double bonds in the phospholipid. Figure 3 is a plot of the number of total double bonds in the sn-1 and sn-2 fatty acids against the change in percent contribution to total PC or PE after TLC or HPLC. The percent change of molecular species in each double bond group, e.g., 0-, 1-, 2-, or 3-double bond(s), etc., were summed and the results are shown in Fig. 3. The net contribution of species having 0 to 3 double bonds was reduced after chromatography. The reduction for PC was 20% after TLC separation and 7.2% after HPLC, while PE lost 19.7% after TLC separation and 7.5% after HPLC. GPLs having a total of 4 to 7 double bonds had a proportionally greater contribution to the total after separation. During the analyses it was apparent, particularly with PCs, that among samples the ion intensities would vary up to 20-fold. Reduction in the total precursor ion intensity correlated with a change in the apparent distribution of PC molecular species in the ES-MS1 spectrum as compared with the distribution in the precursor ion spectrum. The ES-MS1 spectrum contained ions that had masses 22 Da greater than in those in the precursor ion spectrum. A mass increase of 22 mass units suggested replacement of a proton by the sodium cation. We confirmed that sodium adducts are formed by adding sodium acetate to PC standards. Sodiated and unsodiated PC yield a different distribution of product ions as shown in Fig. 4B and C. Protonated PC showed only a strong m/z 184 ion, Fig. 4B. The intensity of the m/z 184 ion from the sodiated PC (Fig. 4C) was substantially reduced relative to other ions. The spectrum of sodiated PC was similar to that reported by Han and Gross (25Han X. Gross R.W. Structural determination of lysophospholipid regioisomers by electrospray ionization tandem mass spectrometry.J. Am. Chem. Soc. 1996; 118: 451-457Google Scholar). The neutral loss of 59 Da (M + Na − 59), m/z 723 in Fig. 4C, is characteristic of the sodiated PC. We now use the 59 Da neutral loss spectrum to ascertain the presence of sodium adducts in PC preparations (25Han X. Gross R.W. Structural determination of lysophospholipid regioisomers by electrospray ionization tandem mass spectrometry.J. Am. Chem. Soc. 1996; 118: 451-457Google Scholar). Sodium adducts can be prevented by treating PC samples dissolved in methylene chloride with 10 μl of formic acid, washing them with water, then diluting the recovered organic layer 1:2 with methanol and adding 5 μl formic acid. Analysis of samples treated in this fashion did not contain sodium adducts (data not shown). Preliminary studies of PC isolated from TLC plates occasionally showed the presence of lower molecular mass PCs that were not present in the unchromatographed extract. Fig. 5A and B show the m/z 184 precursor ion scans before and after TLC. The TLC plate was allowed to stand for an additional 30 min after drying to accentuate oxidation. To exaggerate the conversion and prepare sufficient product for chemical analysis, developed TLC plates were left in a fume hood overnight. Comparing Fig. 6A to that of lipid extract not subjected to TLC (Fig. 6B) suggests that most of the original PC was converted into lower molecular mass PCs. A multiple-step oxidation mechanism was suspected. This mechanism results in the loss of a significant mass of a fatty acid chain and the addition of an oxo-moiety. To establish the presence of an oxo-moiety, the lower molecular mass PCs were either reduced with NaBH4 or converted into a 2,4-dinitrophenylhydrazone. Reduction with NaBH4 gave new PCs having a mass 2 units higher than the starting PC, e.g., the mass of the predominant oxidized component was shifted to m/z 652 (Fig. 6C). Treatment with 2,4-dinitrophenylhydrazine gave PCs having masses 180 units greater than the underivatizedcompounds, e.g., the mass of the predominant oxidized product shifted to m/z 830 (Fig. 6D).Fig. 6.Oxidation of unsaturated PC. Whole cell extract from MDA-231 cells was completely oxidized as described in Materials and Methods by exposing a resolved TLC plate to air overnight. The PC fraction was extracted from silica gel and analyzed by mass spectrometry. PC molecules were detected by precursor ion analysis for m/z 184. Panel A shows a typical, unoxidized PC profile obtained from PCs that were not separated by TLC. 1-O-hexadecanoyl-2-O-(9-octadecadecenoyl)-sn-glycero-3-phosphocholine, m/z 760, is the major molecular species in this sample. Panel B shows the PC precursor ion profile from PCs that were left on a TLC plate for 12 h after development. A glycerophosphocholine with m/z 650 is the predominant molecular species in this sample. Panel C shows the PC profile after reducing the material in Panel B with NaBH4 to form alcohols. The masses of the resultant alcohols are 2 Da higher than their corresponding aldehydes, e.g., m/z 650 shifts to m/z 652. Panel D shows the PC profile generated by treating the material from Panel B with 2,4-dinitrophenylhydrazine. The formation of a 2,4-dintrophenylhydrazone derivative will increase the mass by 180 Da, e.g., m/z 650 shifts to m/z 830. The results are representative of two separate experiments.View Large Image Figure ViewerDownload (PPT) To further characterize the composition of oxidized PCs, we carried out product ion analysis of selected PCs. This procedure uses negative ion detection of the carboxylate anions generated from the demethylated anion [M-15]− (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 product ion analysis of the negative ion m/z 634, which corresponds to the protonated ion m/z 650, gave an anion m/z 171 at sn-2 and m/z 255 at sn-1. Other low molecular mass PCs had positive ion m/z values of 594, 622, 678, and 690. Ion m/z 594, which corresponds to the m/z 578 ion in the negative ion mode, yielded product anions of m/z 115 and m/z 255. To assess the location of sample GPL on a TLC plate after the plate has been developed, phospholipid standards were run in parallel on a separate TLC plate and then stained. It has been suggested that the migration of phospholipidsubspecies within a TLC band may differ from the migration of a single, pure standard. We used ESI-MS/MS to determine whether sample bands of phospholipid classes were homogeneous or if separation of molecular species had taken place. Two different activated TLC silica gel H plates were spotted with egg PC and the plates developed. One of the plates was stained with iodine vapor to visualize PC and the corresponding region of the second TLC plate band was divided into six 1-cm zones. These sections were immediately scraped into separate tubes, extracted, and the GPL analyzed by ESI-MS/MS. Figure 7shows the PC profiles of the six zones. PC subspecies containing 20- and 22-carbon fatty acids migrated at the front of the PC band in fraction 2, while bands 3 through 6 contained almost exclusively shorter chain PCs composed of palmitate and/or stearate. At a collision energy that gives the greatest precursor ion intensity, the sensitivity of PC detection decreases with increasing molecular mass (8Brügger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomo
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