Anal. Chem. 2004, 76, 4515-4521
Automated Monitoring of Phosphatidylcholine Biosyntheses in Plasmodium falciparum by Electrospray Ionization Mass Spectrometry through Stable Isotope Labeling Experiments Christine Enjalbal,*,† Rodolphe Roggero,‡ Rachel Cerdan,‡ Jean Martinez,† Henri Vial,‡ and Jean-Louis Aubagnac†
UMR 5810, Laboratoire des Aminoacides Peptides et Prote´ ines, and UMR 5539, Dynamique Mole´ culaire des Interactions Membranaires, Universite´ Montpellier 2, Place E. Bataillon, 34095 Montpellier Cedex 05, France
The metabolic pathways contributing to phosphatidylcholine biosyntheses in Plasmodium falciparum, the malaria-causing parasite, was explored by electrospray ionization mass spectrometry. Phosphatidylcholine produced by the CDP-choline pathway and by the methylation of phosphatidylethanolamine was identified and quantified through isotopic labeling experiments. A straightforward method based on cone voltage directed in-source fragmentations and relative abundance measurement of endogenous versus deuterated specific fragment ions was developed for simple and rapid automated data acquisition. Such high-throughput analytical protocol allowed us to measure the relative contribution of two different metabolic pathways leading to phosphatidylcholine without performing technically more demanding and timeconsuming MS/MS or LC/MS experiments. Development in the late 1980s of soft ionization techniques such as electrospray ionization1 (ESI) and matrix-assisted laser desorption/ionization2 broadened considerably the scope of mass spectrometry (MS) applications in chemistry and biology. Mass spectrometry is now a key technology for the measurement of both molecular structure and molecular abundances.3 The most accurate quantification is achieved through the use of stable isotope labeling.4 Such MS-based methodology is currently widely used in proteomics5-7 to perform comparative proteome analyses. The original protein and peptide tagging strategies such as isotope* Corresponding author. Tel: (33) 4 67 14 38 19. Fax: (33) 4 67 14 48 66. E-mail:
[email protected]. † UMR 5810, Laboratoire des Aminoacides Peptides et Prote´ines, Universite´s Montpellier 1&2. ‡ UMR 5539, Dynamique Mole´culaire des Interactions Membranaires, Universite´ Montpellier 2. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (2) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (3) Hopfgartner, G.; Bourgogne, E. Mass Spectrom. Rev. 2003, 22, 195-214. (4) De Leenheer, A. P.; Thienpont, L. M. Mass Spectrom. Rev. 1992, 11, 249307. (5) Tao, W. A.; Aebersold, R. Curr. Opin. Biotechnol. 2003, 14, 110-118. (6) Goshe, M. B.; Smith, R. D. Curr. Opin. Biotechnol. 2003, 14, 101-109. (7) Sebastiano, R.; Citterio, A.; Lapadula, M.; Righetti, P. G. Rapid Commun. Mass Spectrom. 2003, 17, 2380-2386. 10.1021/ac049759+ CCC: $27.50 Published on Web 06/17/2004
© 2004 American Chemical Society
coded affinity tag8 and enzymatic digestion9 in H218O are continuously refined to gain in quantification precision.10 Beside protein science, stable isotope labeling combined with mass spectrometry is also used to provide insights into biochemical processes.11 More particularly, cell membranes may be composed of more than 100 phospholipids. Comprehensive determination of their metabolism and molecular species within a phospholipid class has been a challenging task because of the diversity of metabolic pathways and of fatty acid chains and also because of their limited abundance. Although high-performance liquid chromatography (HPLC) allows the separation and quantification of many phospholipid classes, the information concerning the individual molecular species within each class still requires more elaborate techniques and separations. We were interested by using stable isotope labeling combined with mass spectrometry to elucidate metabolic pathways of phospholipid biosynthesis in Plasmodium falciparum, the malaria-causing parasite. Indeed, the aminoglycerophospholipids phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) can be synthesized by multiple pathways.12 The PS pathway encompasses the synthesis of PS, its decarboxylation to PE, and subsequent methylation reactions to form PC. The CDP-choline pathways consist of the synthesis of PE and PC from ethanolamine and choline precursors via cytidine 5′ diphosphate-ethanolamine (CDP-ethanolamine) and CDP-choline intermediates, respectively. The malarial infection caused by P. falciparum, an erythrocyte-invading parasite, is characterized by a dramatic increase in the host cell phospholipid content that reaches 500% at the schizont stage. Of particular interest here is the elucidation of the metabolic pathways that provide the major phospholipid, PC, which constitutes ∼50% of total phospholipids of the intraerythrocytic parasite. As described in Scheme 1, we studied two different pathways leading to PC. The first route, called the CDP-choline pathway, uses choline as precursor whereas the second one, the PE methylation pathway, (8) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (9) Stewart, I. I.; Thomson, T.; Figeys, D. Rapid Commun. Mass Spectrom. 2001, 15, 2456-2465. (10) Lill, J. Mass Spectrom. Rev. 2003, 22, 182-194. (11) Pawlosky, R. J.; Flanagan, V. P.; Novotny, J. A. J. Lipid Res. 2000, 41, 10271031. (12) Dowhan, W. Annu. Rev. Biochem. 1997, 66, 199-232.
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Scheme 1. Structure and Biosynthesis Pathways for the Phosphatidylcholine Phospholipid in P. Falciparum
starts from ethanolamine that is first converted into PE and then methylated to form PC. Each metabolic pathway may represent organelle-specific metabolism, is distinct through its regulation, and may provide molecular species whose diversity is of crucial importance for the cellular functions.13,14 ESI mass spectrometry is so far the best technique to analyze phospholipids.15 Cui et al. have studied deuterium-labeled phosphatidylcholine and phosphatidylethanolamine by electrospray ionization tandem mass spectrometry (ESI-MS/MS) to distinguish between two pathways of phosphatidylcholine biosynthesis.16 Based on these results, we developed a straightforward automated high-throughput method using ESI-MS to qualitatively and quantitatively monitor the biotransformation of choline and ethanolamine into phosphatidylcholine in P. falciparum. MATERIALS AND METHODS Materials. Ethanolamin-d4 and choline-d9 chloride were purchased from Isotec, Inc. (Miamisburg, OH). Ethanolamine, choline chloride, saponin, and phospholipid standards were purchased from Sigma (St. Louis, MO). Modified RPMI 1640 (without choline, serine, methionine, inositol, and folic acid) and Albumax I were purchased from Invitrogen (Cergy Pontoise, France). The human blood and the AB+ human serum were obtained from the local blood bank (Montpellier, France). Incorporation of Deuterium-Labeled Precursors into PC in P. falciparum. The 3D7 strain of P. falciparum was cultured in the presence of basic medium (modified RPMI medium supplemented with 25 mM Hepes, pH 7.4) and 0.5% Albumax I or 10% AB+ human serum and was maintained at 37 °C in modular incubator chambers in a gas mixture of CO2/O2/N2 (5:5:90, v/v/ v).17 Parasite synchronization was obtained with three successive 5% sorbitol treatments.18 Synchronized P. falciparum-infected (13) Vial, H. J.; Ancelin, M. L. Sub-Cellular Biochem. 1992, 18, 259-306. (14) Vial, H.; Ancelin, M. L. In Malaria: Parasite Biology, Biogenesis, Protection; Sherman, I., Ed.; American Association of Microbiology Press: Washington, DC, 1998; pp 159-175. (15) Pulfer, M.; Murphy, R. C. Mass Spectrom. Rev. 2003, 22, 232-364. (16) Delong, C. J.; Shen, Y.-J.; Thomas, M. J.; Cui, Z. J. Biol. Chem. 1999, 274, 29683-29688. (17) Trager, W.; Jensen, J. B. Science 1976, 193, 673-675. (18) Lambros, C.; Vanderberg, J. P. J. Parasitol. 1979, 65, 418-420.
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erythrocytes (0.5% parasitemia, 4% hematocrit, ring stage) were incubated in low-choline medium containing 40 µM choline-d9 chloride and 4 µM ethanolamine-d4 for 5 days. P. falciparuminfected cells (5 × 108 cells) at the trophozoite stage were then collected and washed at 4 °C with 5 mL of cold phosphate-buffered saline. Erythrocytes were then lysed by 0.1% saponin in modified RPMI-1640 during 5 min at 4 °C. After centrifugation at 8000g, the pellet containing the whole mature parasites was stored at -80 °C before being analyzed for lipid content. Extraction of Lipids. Lipids were extracted from the red blood cells according to the method of Folch et al.19 as modified by Ancelin and Vial.20 The chloroformic phase, which contained the deuterated phospholipids, was separated from the aqueous phase and was washed twice with 1.25 mL of chloroform/ methanol/water (3:48:47, v/v/v). The organic phase was then dried under a stream of N2, and the dried materials were dissolved in 200 µL of chloroform/methanol (2:1, v/v) and analyzed by mass spectrometry. Mass Spectrometry. ESI mass spectra were recorded on a QTof I mass spectrometer (Waters-Micromass, Manchester, U.K.) fitted with a Z-spray ionization source. Calibration was performed by an aqueous solution of phosphoric acid (0,1%) in the mass range of 50-1000 Da. Nitrogen was used as both nebulizing and drying gas. Temperatures of the source and of the drying gas were set at 80 and 150 °C, respectively. The capillary voltage was set at 2900 V whereas the cone voltage was fixed at 30 or 90 V. Argon was chosen as collision gas in MS/MS experiments. An HPLC module Waters 2790 equipped with an autosampler and a photodiode array ultraviolet (UV) detector PDA 996 (Waters-Micromass) was coupled to the mass spectrometer. Elution was performed in the HPLC module at a flow rate of 0.6 mL/min under isocratic conditions, 25% solvent A (water + 0.1% trifluoroacetic acid) and 75% solvent B (acetonitrile + 0.1% trifluoroacetic acid). Samples were injected every minute. The flow was split prior to the mass spectrometer entrance allowing roughly 1/10th of the eluent in the ESI source. The MassLynx 4.0 software (WatersMicromass) controlled all pieces of equipment. All samples were made by aliquoting 25 µL of the lipidic extract solution, which was then diluted with 0.5 mL of a water/acetonitrile solution supplemented with 0.1% trifluoroacetic acid (25:75, v/v). A 250µL aliquot was injected for each experiment. RESULTS AND DISCUSSION The general formulas of the studied phospholipids are given in Scheme 1. Molecular diversity is provided by the variation of three substructures: the polar head (R) that defines the phospholipid class and the two acyl chains (R1 and R2) that constitute the lipophilic part of the molecule. Among all phospholipid classes, the nitrogen-containing molecules such as PCs and PEs yield abundant protonated molecular ions in ESI mass spectrometry under positive ionization conditions.21 Besides, PC and PE constitute the only phospholipid classes exhibiting a net neutral charge, the negative charge of the phosphate group being neutralized by the positive charge of the quaternary ammonium (Scheme 1). (19) Folch, J.; Lees, M.; Sloane Stanley, G. H. J. Biol Chem. 1957, 226, 497709. (20) Ancelin, M.; Vial, H. J. Biochim. Biophys. Acta 1989, 1001, 82-89. (21) Fang, J.; Barcelona, M. J. J. Microbiol. Methods 1998, 33, 23-35.
Figure 1. Mass spectra of lipidic extracts recorded at different cone voltage values. (A) Mass spectrum recorded at a low cone voltage value (30 V) allowing detection of intact phospholipids (protonated molecules between m/z 670-850); (B) mass spectrum recorded at a high cone voltage value (90 V) inducing phospholipid fragmentations (protonated molecules between m/z 670 and 850, fragment ions between m/z 450 and 650 issued from protonated PE molecules, and the most abundant fragment ion at m/z 184 produced from all protonated PCs).
Deciphering the metabolic pathways of phosphatidylcholine biosynthesis in P. falciparum required the following studies, which will be discussed in detail below. First, the chloroform lipidic extract needed to be analyzed by ESI-MS to visualize all protonated molecules. The class of each targeted phospholipid (PC and PE) was then evidenced according to specific fragmentation patterns obtained under collisionactivated dissociation (CAD) conditions in MS/MS experiments. We did not try to fully characterize each detected PE and PC by determining the nature of the acyl chains, but we concentrated our efforts on establishing which metabolic pathways was providing the bulk of the new PC molecules. For that purpose, deuterium was used as a stable tag to distinguish the biotransformations of ethanolamine and choline into PC species. Specific mass shifts according to the metabolic pathway were expected. Since such shifts were not clearly seen on the recorded ESI mass spectra, determination and relative quantification of the PC biosynthesis routes were performed by in-source CAD experiments through the detection of deuteriumlabeled fragment ions and the direct measurement of their relative abundances on the mass spectra. Finally, method reproducibility was validated through intraand interday analyses. Automated data acquisition provided highthroughput phospholipid extract characterization. MS Characterization of the Phospholipids Produced by P. falciparum. (a) Sample Workup: From Phospholipid Extract to Mass Spectrometry Analysis. Phospholipid fractions were recovered as a 10-5 mol/L solution in chloroform/methanol (2:1), which
is not suitable for direct ESI-MS analysis that requires aqueousbased samples. Great caution was taken during sample preparation due to the very poor solubility of phospholipids in water-containing media. An optimized ESI-MS protocol was set up involving dilution of the chloroform/methanol extract solution with acetonitrile in acidic water. The percentage of water drastically affected the analysis outcome whereas the nature of the acidic additive (acetic acid, formic acid, or trifluoroacetic acid) did not interfere. Reliable sensitive analyses were achieved with a 25% final water content ensuring efficient MS detection. Highly reproducible positive ion mass spectra were recorded for the same lipidic extract analyzed on different days or for various lipidic extracts characterized on the same day. A typical positive ion ESI mass spectrum is reproduced in Figure 1A. The tuning of the cone voltage value that is responsible for ion extraction from the ionization source into the mass analyzer affects the analysis result. Low cone voltage values allowed to record only protonated molecules (for instance, 30 V in Figure 1A). The protonated phospholipid molecules were detected in the mass range of 670-850 Da. Ions of lower molecular masses were attributed to solvent adducts and sample impurities (m/z 140-500). High cone voltage values (for instance, 90 V in Figure 1B) induced some in-source dissociations leading to the presence of both protonated phospholipid molecules (m/z 670850) and fragment ions (m/z 184 and 450-650) in the mass spectrum. Structural elucidation of these phospholipids was undertaken by positive ion ESI-MS/MS to reveal the nature of the polar head. Analytical Chemistry, Vol. 76, No. 15, August 1, 2004
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Scheme 2. Collision-Activated Dissociation (CAD) of Protonated PE and PC Molecules in MS/MS Experiments
(b) Fragmentation Patterns of PC and PE in Positive MS/MS Experiments. MS/MS experiments aim to dissociate parent ions upon energetic collisions with an inert gas into fragment ions. The general fragmentation pattern upon such collision-activated dissociation conditions is as follows: m1+ f m2+ + Neutral molecule. The parent ion (m1+) is selected by the first mass analyzer and is broken inside the collision cell into a fragment ion (m2+) and a neutral molecule. Only the fragment ion is detected by the second mass analyzer. Fragmentation mechanisms of PC and PE established in MS/ MS experiments on triple quadrupole instruments22,23 (QqQ) were validated on an hybrid mass spectrometer possessing a time-offlight (QqTof) mass analyzer. As expected from literature data,22 phosphatidylethanolamine loses its polar head as a neutral molecule of phosphoethanolamine weighing 141 Da (Scheme 2). Upon positive ESI ionization, protonation of the amino group occurred; the proton moved then onto an oxygen of the lipophilic chain and triggered the phosphoethanolamine loss. Such loss of 141 Da was observed for any PE species since the variable positions, R1 and R2, remained on the detected fragment ion. Thus, the value of any PE fragment ion varied according to the nature of the alkyl substituents R1 and R2. On the contrary, phosphatidylcholine that possessed a trimethyl quaternary ammonium moiety kept the charge on the polar head during positive ESI ionization. Upon CAD conditions, the fragmentation mechanism involves an hydrogen transfer from the R2 chain onto the phosphate group and the subsequent release of phosphocholine23 as shown in Scheme 2. The resulting fragment ion was detected at m/z 184 (Figure 1B). Thus, the same phosphocholine ion was produced from all protonated PC molecules whatever the nature of the R1 and R2 acyl chains. (22) Kerwin, J. L.; Tuininga, A. R.; Ericsson, L. H. J. Lipid Res. 1994, 35, 11021114. (23) Hsu, F. F.; Turk, J. J. Am. Soc. Mass Spectrom. 2003, 14, 352-363.
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Each detected phospholipid in the ESI mass spectrum of the cellular lipidic extract (Figure 2A) was subjected to MS/MS experiment (Figure 2C and E). Most of them were attributed to PC. For instance, in the mass spectrum displayed in Figure 2A, the ions at m/z 703.6, 734.6, 746.7, 758.6, 760.7, 786.7, 788.7, and 813.8 were protonated PC molecules whereas the ion at m/z 718.6 was attributed to PE. Quantification of the detected PC directly from the recorded ESI mass spectrum was prevented due to differences in phospholipid ionization efficiency. Indeed, model solutions containing four commercial PCs at known concentrations exhibited distorded protonated ion abundances, one molecule being overexpressed (Supporting Information Figure S-1). The respective amounts of the recorded phospholipids produced in P. falciparum were thus measured by stable isotope tagging methodology. Determination of PC Biosynthesis in P. falciparum. (a) Stable Isotope Labeling Experiments. The targeted biotransformations to be evaluated are described in Scheme 1. The CDP-choline pathway represents the major route in competition with an alternative path, the PE methylation pathway. One way to differentiate these routes was to feed cells with deuterated precursors in the form of choline-d9 and ethanolamine-d4 leading to deuteriumlabeled PC (Scheme 3): PC-d9 and PC-d4 for phospholipids issued from choline and from ethanolamine, respectively. Mass shifts of 9 and 4 Da were expected from each detected protonated molecule in positive ESI mass spectrometry. However, only the most abundant species exhibited a weak d9-tagged ion. For instance, the most abundant protonated PC at m/z 760 provided upon labeling a noticeable ion at m/z 769 (Figure 2B and D), but the corresponding tagged ion issued from the PE methylation pathway at m/z 764 was not abundant enough to be observed (Figure 2B). So, deuterium incorporation did not occur in sufficient yield to allow direct PC biosynthesis elucidation from the primary ESI
Figure 2. Lipidic extract mass spectra. (A) Data recorded at 30 V displaying only the range of m/z 640-880 that contained the protonated phospholipid molecules. The infected erythrocytes were incubated for 5 days without any deuterated precursors (see Figure 1A for complete mass spectrum over m/z 100-1000). (B) Data recorded at 30 V. The infected erythrocytes were incubated for 5 days in the presence of cholined9 and ethanolamine-d4 at a physiological concentration of 40 and 4 µmol/L, respectively. Only two PC-d9 were clearly seen at m/z 769.7 and 797.8. (C) CAD mass spectrum recorded in MS/MS experiments by dissociation of the protonated molecular ion at m/z 760 giving the fragment ion at m/z 184 indicating a PC molecule. (D) CAD of m/z 769 giving the fragment ion at m/z 193 indicating a PC-d9 molecule. (E) CAD of m/z 718 giving the fragment ion at m/z 577 (loss of the neutral phosphoethanolamine at 141 Da) indicating a PE molecule.
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Scheme 3. PC Biosynthesis Pathways in P. Falciparum in the Presence of Deuterated Precursors
Table 1. Relative abundances of Endogenous and Labeled Polar Head Fragment Ions from a Lipidic Extract Injected 10 times in a Row (Incubation in Albumax in the Presence of Choline-d9 and Ethanolamine-d4 at Physiological Concentrations of 40 and 4 µmol/L, Respectively) relative abundances (%) of polar head fragment ions from injection number exp
iona
1
2
3
4
5
6
7
8
9
10
average (%)
SD
1
184 188 193 184 188 193
27.6 5.8 66.6 22.2 13.9 63.9
29.7 8.1 62.2 28.6 5.7 65.7
21.9 6.2 71.9 26.3 13.1 60.6
21.9 6.2 71.9 30.7 10.3 59.0
29.7 8.1 62.2 26.3 13.1 60.6
21.2 9.1 69.7 42.6 8.5 48.9
39.1 10.9 50.0 27.8 8.3 63.9
27.8 8.3 63.9 27.8 8.3 63.9
24.2 6.1 69.7 34.2 5.3 60.5
24.2 6.1 69.7 28.9 10.5 60.6
26,7 7,5 65,8 29,5 9,7 60,8
5,4 1,7 6,7 5,5 3,0 4,7
2
a
m/z 184, polar head fragment ion of PC-d0; m/z 188 polar head fragment ion of PC-d4; m/z 193, polar head fragment ion of PC-d9.
mass spectra. Only MS/MS experiments allowed to perform the desired studies. (b) Stable Isotope Labeling MS/MS Experiments. Collision and subsequent dissociation of the protonated PC molecules were performed in the initial stage of the mass spectrometer (source transport region) prior to the mass analyzer, which can be of any type (Q, Tof, ion trap, etc.). This MS method is thus universal and works with standard MS equipment fitted with only one mass analyzer. Two cone voltages were set up: a low cone voltage (30 V) to acquire the ESI mass spectra and a high cone voltage (90 V) to induce in-source CAD fragmentations. Any PC molecule were fragmented and led to the polar head at m/z 184 when the parent ion was not deuterated, at m/z 193 when the parent ion incorporated nine deuterium atoms (CDP-choline pathway), or at m/z 188 when the parent ion incorporated four deuterium atoms (PE methylation pathway) (Scheme 3). As stated previously, deuterium incorporation was not clearly observed in MS analyses (Figure 2A and B). On the contrary, CAD experiments provided abundant deuterium-containing ions. Such results came from the 4520 Analytical Chemistry, Vol. 76, No. 15, August 1, 2004
fact that the targeted labeled phosphocholine fragment ion was produced from the fragmentation of all PCs present in the lipidic extract. In other words, all fragment ions issued from all PC-d0 were cumulated under a single ion (m/z 184). Similarly, CAD of any PC-d4 and PC-d9 molecules produced only one ion at m/z 188 and 193, respectively. The fact that such a fragment ion (m/z 184, 188 or 193) was issued from several molecules explains the sensitivity of its detection. The ratio between this two deuteriumlabeled ions (m/z 188 and 193) defines the relative contribution of the CDP-choline pathway and PE methylation pathway to the synthesis of new phospholipid molecules (Scheme 3). As depicted in Scheme 3, there was some native phosphatidylcholine and phosphatidylethanolamine in the cell originating from the host erythrocyte membrane explaining why nonlabeled protonated molecules were still observed on the primary mass spectra (Figure 2B) and nonlabeled fragment ion at m/z 184 in CAD mass spectra (Supporting Information S-2). Under these conditions, only relative quantification was possible, the nonlabeled fragment ion (m/z 184) acting as an internal standard.
(c) Relative Quantification of PC Biosynthesis Pathways. Ion suppression during competitive ionization and versatile ion transmission from the source into the mass analyzer may affect the relative ion abundance measurement accuracy. The first difficulty was avoided by the use of isotopic labeling, the hydrogenand deuterium-containing molecules behaving similarly during the ionization process.5 To average ion transmission, each sample was injected 10 times in a row under automated flow injection analysis conditions. Each analysis lasted 1 min, including injection cycle and MS data acquisition, allowing automated high-throughput analyses. Relative abundances of m/z 184, 188, and 193 ions were measured on the mass spectra and converted into percentages (Table 1 and Supporting Information S-3). The standard deviation values were calculated and were less than 7%. No injection was discarded even if one in each experiment was obviously out of range (injection 7 in experiment 1, and injection 6 in experiment 2). Based on an average of two independent experiments (experiments 1 and 2 in Table 1), each carried out in triplicate, there was 28% of remaining nonlabeled PC, 63% of PC produced from the CDP-choline route, and 9% of PC coming from the PE methylation path. Taking the nonlabeled molecule as a reference, that is the ion at m/z 184 as an internal standard, the ratio PCd9/PC-d4 equaled to 7.6. Thus, the two PC biosynthetic routes were estimated around 88% for the CDP-choline path and 12% for the PE methylation pathway. In the search of an absolute quantification method, we designed and synthesized an appropriate phosphatidylcholine to mimick at best the MS behavior of endogenous and deuterated PC molecules. A mass increment of 14 Da was introduced by N-alkylation with ethyl iodide of commercial N,N-dimethylphosphatidylethanolamine. As expected, the MS/MS experiment provided for such standard molecule a fragment ion at m/z 198 (Supporting Information Figure S-4). Surprisingly, the analysis of model mixtures containing the standard and endogeneous PC revealed great variations in ion abundances, the standard polar head (m/z 198) being overexpressed compared to the PC polar head (m/z 184) (Supporting Information Figure S-5). So, absolute quantification from ion abundances of two homologated molecules would not be reliable.
From this experiment, the respective abundances of PC-d0 (57%), PC-d4 (5%), and PC-d9 (38%) were directly compared without taking into account the standard abundance. Even when less deuterated materials were produced (57% of remaining PC-d0 compared to 28% in the previous experiment), the relative ratio of PC-d9/PC-d4 was identical to the one previously determined (7.6 in all experiments; i.e., 88% of the produced PC came from the CDP-choline route and 12% from the PE methylation pathway), demonstrating the accuracy and reproducibility of the developed MS-based method. CONCLUSION Under high-throughput loop injection analyses, we found that PC species exhibited different ionization efficiencies in ESI mass spectrometry that could not be correlated to their molecular weights. So, quantification of isotopically labeled PC (PC-d0, PCd4, and PC-d9) was performed from straightforward automated insource CAD experiments. The production of a single fragment ion issued from all PC molecules alleviated the lipidic extract complexity. Whatever the number and relative quantities of the PC species, one abundant probe ion (polar head fragment ion) was generated, enabling sensitive analyses on a restricted mass range. Direct measurement of the relative deuterated polar head fragment ion abundances allowed us to determine with high accuracy and reproducibility the distinct contribution of the CDPcholine pathway or the PE methylation pathway to the bulk of cellular newly synthesized PC. ACKNOWLEDGMENT We thank Pr. M. Calas, Dr. K. Alarcon, and Dr. C. Carcel for the synthesis of the N,N,N-ethyldimethylphosphatidylethanolamine compound. SUPPORTING INFORMATION AVAILABLE Additional informations as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 12, 2004. Accepted May 12, 2004. AC049759+
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