Proc. Natd. Acad. Sci. USAVol. 91, pp. 10635-10639, October 1994Chemistry

Electrospray ionization mass spectroscopic analysis of humanerythrocyte plasma membrane phospholipids

(plasaogen/mass sectrometry/sph ye l/red blood cell)

XIANLIN HAN AND RICHARD W. GROSSDivision of Bioorganic Chemistry and Molecular Pharmacology, Departments of Internal Medicine, Chemistry and Molecular Biology and Pharmacology,Washington University School of Medicine, St. Louis, MO 63110

Communicated by S. I. Weissman, July 11, 1994 (received for review July 28, 1994)

ABSTRACT Electrospray ionization mass spectrometry(ESI-MS) was utilized for the structural determination andqmntitative analysis of individual phospholipid molecula spe-des from subplcomole amounts of human erythrocyte plasmamembrane phospholipids. The sensitivity of ESI-MS was 2-3orders of magnitude greater than that achievable with fast-atom bombardment mass sectrometry (FAB-MS). Phospho-lipid structure determination and quantitative analysis withESI-MS can be performed directly from chloroform extracts ofbiologic samples, obviating the need for prior chromatographicseparation of phospholipid classes which has been inFAB-MS phospholipid analyses. Furthermore, ESI-MS is un-complicated by differential frgentation of molecular ionsand idiosyncratic surface desorption, allowing the quantitationof phospholipids with coefficients of determination (r2) > O.99and accuracies > 95%. More than 50 human erythrocyteplasma membrane phospholipld constituents were identified bydirect ESI-MS analysis of chloroform extracts of plasma mem-branes derived from the equivalent of <1 id of whole blood.The majlor ethanolamine glycerophospholipid subclass inerythrocyte plas membranes ws plasmenyeth a ethat was highly enriched in polyunsaturated fatty acids at thesn-2 position. Collectively, these results demonstrate thatESI-MS of phospholpds is an enabling strategy for the directstructural deternai on and quantitative analysis of subpco-mole amounts of phospholipids from biologic samples.

During the last decade neutral-atom collisional activation ofanalytic targets [e.g., Xe or Cs fast-atom bombardment massspectrometry (FAB-MS)] has opened new dimensions in theanalysis of a variety of phospholipids derived from syntheticprocesses or biologic sources (1-4). However, the differentialvolatilization of chemically distinct phospholipids resultingfrom small alterations in their surface properties, coupledwith the modest ionization efficiency inherent in FAB-MS,has collectively limited the utility of this technique for thequantitative analysis of phospholipid classes and subclassesfrom biologic sources where diminutive amounts of materialare available. Furthermore, the high-energy ionization inher-ent in the FAB-MS technique, in conjunction with thechemical complexity and lability of biologic phospholipids,results in substantial and differential fragmentation rates ofmolecular ions from individual phospholipid classes andsubclasses volatilized by FAB, thereby further confoundingprecise quantitative analysis (5-9). Progress has been madeusing laser desorption ionization for phospholipid analysis,but the presence of complicated quasimolecular ions andfragmentation of those ions has precluded precise quantita-tion (10-12).

It is now apparent that cellular activation is mediated, inlarge part, through the generation of lipid second messengers

resulting from the activation of a multiplicity of differentphospholipases (13-16). Accordingly, identification of thetemporal course and extent of hydrolysis of specific phos-pholipid molecular species during cellular activation is aprominent question in signal transduction research. How-ever, difficulties inherent in the structure determination andquantitation of subpicomole amounts of zwitterionic phos-pholipids present in biologic samples have thus far precludedfacile analyses. Prior studies have demonstrated that elec-trospray ionization mass spectrometry (ESI-MS) allows ac-cess to a vast array of heretofore inaccessible problems inprotein chemistry through dramatically enhanced volatiliza-tion of molecular ions (17-20). However, to date, the utilityof ESI-MS for the structure identification and quantitativeanalysis of phospholipids present in biologic membranes hasnot been realized. We now report that ESI facilitates thestructural determination and quantitative analyses of indi-vidual molecular species of synthetic and naturally derivedphospholipids with a sensitivity 2 to 3 orders of magnitudegreater than that previously achieved by FAB-MS. Since thisionization modality is not complicated by differential frag-mentation and idiosyncratic surface desorption, it will allowthe direct quantitation and structural analysis ofphospholip-ids from extracts of biologic samples.

MATERIALS AND METHODSMaterials. Commercially available phospholipids were

purchased from Avanti Polar Lipids or Nu Chek Prep (Ely-sian, MN). 1-O-(Z)-Hexadec-l'-enyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine were prepared as de-scribed (21). All phospholipids and fatty acids werequantitated by capillary gas chromatography after acid meth-anolysis (22).ESI-MS. ESI mass spectra were acquired on a triple-

quadrupole tandem mass spectrometer (Finnigan MAT TSQ700, San Jose, CA) equipped with an electrospray interface(Analytica of Branford, Branford, CT). The first and thirdquadrupoles of the instrument serve as independent analyz-ers and the second is a nonlinear octapole, which is utilizedas a collision cell for MS-MS experiments. The detector ofthe instrument is an off-axis continuous dynode electronmultiplier operable from -400 to -3000 V, with a variablepostacceleration/conversion dynode voltage from -3 kV to-20 kV or +3 kV to +20 kV for detection of positive ornegative ions, respectively. The effective mass range wasfrom m/z 10 to m/z 4000 with mass peak position stable to

Abbreviations: FAB, fast-atom bombardment; ESI, electrosprayionization; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine;POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; POPA,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol.

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A B

7.0

6.5

._

c

a)

01)

800m/z

-2 -1 01600

log(DPPC) (pmol)

FIG. 1. Concentration dependence of positive-ion ESI massspectra of DPPC. (A) A positive-ion ESI mass spectrum of DPPC(0.01 pmol/t4) obtained during a 1-min infusion at a flow rate of 1pl/min (i.e., 0.01 pmol consumed) shows a single molecular ion atm/z 757 ([M+Na]+) with a signal/noise ratio of 4. (B) The doublelogarithmic plot of the signal intensity (i.e., ion counts) vs. theamount of DPPC consumed yielded a line with a slope = 1.006 +0.005 and a y intercept = 6.094 ± 0.008. The sensitivity of the ESImethod is illustrated by the absolute value of the slope [(1.242 +0.0023) x 106 ion counts/pmol] and the accuracy is illustrated by thecoefficient of determination (r2 = 0.9987).

0.05 unit over 8 hr. The electrospray ion source and itsfunctional parts have been described in detail (23). For allexperiments, both the electrospray needle and the skimmerwere operated at ground potential, whereas the electrospraychamber (i.e., cylindrical electrode) and metallized entranceof the glass capillary were operated at -3.5 kV in thepositive-ion mode and at +3.0 kV in the negative-ion mode.A positive or negative 90-V potential was applied to themetallized exit of the glass capillary for positive- or negative-ion spectra, respectively. A separate positive or negative175-V potential was placed on the tube lens for acquisition ofpositive or negative ions, respectively. The temperature ofnitrogen drying gas as it entered the electrospray chamberwas set at 100°C and the drying gas was held at a constantpressure of25 psi (1 psi = 6.89 kPa). Typically, a 1-min periodof signal averaging was employed for each spectrum. All

A

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samples were dissolved in 1:2 chloroform/methanol andinfused directly into the ESI chamber with a syringe pump ata flow rate of 1-2 gl/min.

Preparation of Human Erythrocyte Plasma Membranes andESI-MS. Plasma membranes derived from human erythro-cytes were prepared as described (24). Erythrocyte plasmamembranes were resuspended in 5 mM sodium phosphatebuffer (pH 8) and were extracted by the Bligh-Dyer method(25). Samples for ESI-MS analyses were evaporated under astream of nitrogen and the residue was resuspended in 30 plof 1:2 chloroform/methanol in the presence of a modestmolar excess of NaOH (typically 1-10 ttM). Resuspendedsample was directly utilized for acquisition of positive-ionand negative-ion ESI mass spectra. Tandem MS after ESIwas performed similarly to that previously described afterFAB ionization (26, 27), and the detailed descriptions of thepathways leading to the observed product ions will be pre-sented elsewhere. Choline and ethanolamine glycerophos-pholipids were separated on an Ultrasphere-Si column (4.5mm x 250 mm; 5-pm particle size; Beckman) (28) prior toquantitation by capillary gas chromatography (22).Data Analysis. For correction of 13C isotope effects, sum-

marization of nonisotopic and isotopic peak intensities andsubsequent normalization to the lowest mass molecular spe-cies were used. The peak intensities after correction for the13C isotope effect are presented with horizontal bars.

RESULTS AND DISCUSSIONTo determine the utility of ESI for the mass spectrometricanalysis of phospholipids, initial experiments examined theconcentration dependence of positive-ion ESI mass spectraof 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)(Fig. 1). The positive-ion ESI mass spectra of DPPC duringinfusion of a 0.01 pmol/Al solution into the ionization cham-ber at a flow rate of 1 A4/min, utilizing a 1-min signal-averaging interval (i.e., 0.01 pmol consumed), demonstrateda single peak at m/z 757 ([M+Na]+) with a signal/noise ratioof4 (Fig. 1A). No multiply charged ions were observed underthe experimental conditions employed. Because continuous-flow FAB-MS (the most sensitive FAB method) consumes 3pmol ofDPPC to obtain a similar signal/noise ratio (29), theseresults demonstrate the 300-fold improvement in the detec-tion limit of ESI-MS compared with FAB-MS. The correla-tion between the mass of DPPC and the sodiated DPPC ion(i.e., [M+Na]+) peak intensity was linear over a dynamicrange of 1000 (from 0.01 to 10 pmol) with a slope = (1.242 ±

B[POPE - H]-

717100

80 FIG. 2. ESI analyses of the major phospho-lipid classes can be selectively accomplished byutilization of either positive- or negative-ion

-60 spectral modes. (A) A positive-ion ESI massspectrum of an equimolar mixture of 1-palmi-toyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC, m/z 783) and 1-palmitoyl-2-oleoyl-sn-

40 glycero-3-phosphoethanolamine (POPE, m/z[POPE + Na]+ [POPC + OH]- 741) (5 pmol/pl each) demonstrates the prepon-

741 777 derance of the choline glycerophospholipid20 \ [POPC + Cl]- peak in this ion mode. (B) A negative-ion ESI

0795 mass spectrum of an equimolar mixture (5pmol/pl each) ofPOPE (m/z 717, [M-H]-) andPOPC (m/z 795, [M+CI]-; m/z 777, [M+OH]-)in the presence ofNaOH (10 pmol/j4) illustrates

650 700 750 800 850 200 400 600 800 1000 the preponderance ofthe POPE molecular ion inm/z m/z this ion mode.

10636 Chemistry: Han and Gross

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0.0023) x 106 ion counts/pmol and a coefficient of determi-nation (r2) = 0.9987 (Fig. 1B). With large increases in DPPCconcentration, multiply charged ions and aggregated ions(e.g., [3M+2Na]2+ and [2M+Na]+) were detected and grad-ually became dominant at concentrations > 100 ,uM. Whenthe pH of the infused DPPC solution was decreased, theprotonated DPPC ion (i.e., [M+H]+) was detected withsimilar sensitivity. Furthermore, although FAB-MS of phos-phatidylserine results in multiple fragment ions (e.g., >80%fragmentation is present in phosphatidylserine analysis usingFAB-MS) (4, 5), the negative-ion ESI mass spectrum of1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)(0.01 pmol/pl) contained a single molecular ion peak (m/z761, [M-HV-) with a signal/noise ratio of 8 during a 1-mininfusion at 1 td/min (i.e., 0.01 pmol consumed) (data notshown). An [M-2H]2- ion of phosphatidylserine was alsodetected with either marked increases in the concentration ofthe phosphatidylserine in the infused solution or with in-creases in the pH of the infused solution. A linear correlationbetween the mass of POPS consumed and its molecular ions(i.e., [M-H]- and [M-2H]2-) was present with a slope =(1.18 ± 0.04) x 106 ion counts/pmol and r2 = 0.9993 over a10,000-fold dynamic range (0.01-100 pmol consumed).Many classes of phospholipids possess net negative charge

at neutral pH. Accordingly, negative-ion ESI mass spectra ofthese phospholipids can be efficiently obtained with [M-H]-as the molecular ion peak (see above). However, phosphati-dylcholine, phosphatidylethanolamine, and sphingomyelinare zwitterionic molecules, and therefore either positive- ornegative-ion mass spectra of these phospholipid classes areaccessible through ESI-MS. Negative-ion ESI mass spectraof ethanolamine glycerophospholipids contain only the[M-H]- ion and are far more sensitive than positive-ionspectra, due to the facile loss of a proton from the ammoniumion in ethanolamine glycerophospholipids. As anticipated,exposure to base further enhances the sensitivity of negative-ion ESI-MS of ethanolamine glycerophospholipids. In con-trast, choline glycerophospholipids and sphingomyelins aremore efficiently analyzed in the positive-ion mode as thesodiated ion complex under normal analysis conditions.Thus, positive-ion ESI mass spectra of equimolar mixtures ofcholine and ethanolamine glycerophospholipids predomi-nantly demonstrate [M+Na]+ ions of choline-containingphospholipids (Fig. 2A), whereas negative-ion ESI massspectra of equimolar mixtures predominantly demonstrate[M-H]- ions of ethanolamine glycerophospholipids (Fig.2B).The extraordinary sensitivity and selectivity of ESI-MS for

phospholipid analysis in both the negative-ion and the positive-ion mode were exploited to directly identify the structure andmole fraction of individual phospholipid constituents fromchloroform extracts of human erythrocyte plasma membraneswithout prior chromatographic separation of phospholipidclasses. From the equivalent of only 25 nl of whole blood (13.5pmol of choline glycerophospholipids), a positive-ion ESI massspectrum was obtained which demonstrated multiple molecularspecies of choline glycerophospholipids (e.g., m/z 757, 781,783, 805, 809, and 833, corresponding to sodiated 16:0-16:0,16:0-18:2, 16:0-18:1, 16:0-20:4, 18:0-18:2/18:1-18:1, and 18:0-20:4 phosphatidylcholines, respectively, as well as m/z 767,795, and 823, corresponding to sodiated 16:0-18:1, 18:0-18:1,and 18:0-20:1 plasmenylcholines, respectively) (Fig. 3A). Inaddition, multiple molecular species of sphingomyelin weredemonstrated, including species at m/z 726, 754, and 782,corresponding to 16:0, 18:0, and 20:0 amides (Fig. 3A). Anegative-ion ESI mass spectrum ofphospholipid extracts fromthe same sample demonstrated >20 molecular species ofethanolamine glycerophospholipids (i.e., from 11 pmol ofethanolamine glycerophospholipids) (Fig. 3B). Remarkably,human erythrocyte plasma membrane ethanolamine glycero-

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FIG. 3. Direct ESI-MS analysis of human erythrocyte plasmamembrane phospholipids. (A) A positive-ion ESI mass spectrum oferythrocyte plasma membrane phospholipid extract (containing 13.5pmol of choline glycerophospholipids from 25 nl of whole blood)shows 14 molecular species of choline glycerophospholipids and 4molecular species of sphingomyelin. (B) A negative-ion ESI massspectrum of the same extract of plasma membrane phospholipidsshows >25 molecular species of ethanolamine glycerophospholipidsand 8 molecular species of serine and inositol glycerophospholipids.(C) In ESI-MS-MS analysis of isobaric ethanolamine glycerophos-pholipids (m/z 777) extracted from human erythrocyte plasma mem-branes, selection and collisional dissociation of m/z 777 after nega-tive-ion electrospray ionization demonstrated two carboxylic anions(m/z 329 and 331) (see Inset), corresponding to 22:5 and 22:4 fattyacids, respectively. In addition, several ethanolamine lysoglycero-phospholipid-type ions were identified, facilitating the assignment ofthese species as 18:0-22:5 and 18:1-22:4 plasmenylethanolamines ina 2:1 molar ratio. The phospholipid extract of human erythrocyteplasma membrane was prepared as described in Materials andMethods. The sample, in 30 A4 of 1:2 chloroform/methanol in thepresence of excess NaOH (i.e., NaOH/lipid molar ratio > 1), wasinfused directly into the ESI chamber with a syringe pump at a flowrate of 1.5 Al/min for mass analyses in both negative- and positive-ion modes.

phospholipids were predominantly composed of plasmalogenmolecular species, with 1-(0)-Z-octadec-1'-enyl-2-eico-satetra-5' ,8',11',14'-enoyl-sn-glycero-3-phosphoethanola-mine (i.e., 18:0-20:4 plasmenylethanolamine, m/z 751) repre-senting the major individual molecular species (=20 mol%)present. Other plasmenylethanolamines, containing 20:4,22:4, 22:5, and 22:6 fatty acids at the sn-2 position, were alsoidentified (e.g., m/z 723,749,775, 777, and 779, correspondingto 16:0-20:4, 18:1-20:4, 18:0-22:6/18:1-22:5, 18:0-22:5/18:1-22:4, and 18:0-22:4/20:0-20:4 plasmenylethanolamines, re-spectively). In addition, phosphatidylethanolamines at m/z

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AArachidonic POPG

Acid POPA 748 PoPS303 674 71

100 674 7I

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~60675

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670 675 680

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Molarratio 10

748

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a-

0

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717

700 750 700 750 700 750

m/z

C

0 2 4 6 8 10POPG/POPEmolar ratio

FIG. 4. Quantitative analysis of structurally diverse lipids by negative-ion ESI-MS. (A) A negative-ion ESI mass spectrum of an equimolarmixture of arachidonic acid (m/z 303), POPA (m/z 674), POPE (m/z 717), POPG (m/z 748), and POPS (m/z 761 and 380) was obtained in thepresence of excess NaOH (30 pmol/ld). Mixtures containing each compound at 5 pmol/A4 were infused directly into the ionization chamberat 1 pl/min and a 1-min acquisition time was used. Correction for the 13C isotope factor (see Inset for an example) was performed (shown byhorizontal bars on individual phospholipid peaks), demonstrating similar molecular ion intensities (within 5%). (B) Negative-ion ESI massspectroscopy of POPE (10 pmol/0) and POPG (1, 10, and 100 pmol/i4 are shown) binary mixtures was performed to quantify the sensitivityratio of ESI volatilization of POPE to POPG. Samples were infused into the sample chamber at 1 AI/min for 1 min during sample acquisition.(C) Correlation ofthe POPG/POPE molar ratio (seven different binary mixtures) to the ratio oftheir molecular ion peak intensities demonstrateda linear relationship with a slope = 1.50 ± 0.07, and an r2 = 0.998.

715, 717, 739, 765, 767, and 793, corresponding to 16:0-18:2,16:0-18:1, 16:0-20:4, 18:1-20:4, 18:0-20:4/16:0-22:4, and 18:0-22:5/18:1-22:4, respectively, were also demonstrated. Theassignment of aliphatic chains and their regiospecificity ineach molecular species was made through tandem MS of thecorresponding molecular ions (e.g., Fig. 3C). These resultswere substantiated by conventional techniques, including re-verse-phase high-performance liquid chromatography andcapillary gas chromatography of choline and ethanolamineglycerophospholipids as previously described (30). Negative-ion ESI mass spectra of erythrocyte plasma membrane phos-pholipid extracts also demonstrated multiple molecular spe-cies of serine and inositol glycerophospholipids containingpolyunsaturated fatty acids (e.g., m/z 811, 833, and 886,corresponding to 18:0-20:4 phosphatidylserine, 18:1-22:6phosphatidylserine, and 18:0-20:4 phosphatidylinositol) (Fig.3B).

Substantial differences in surface interactions of differentphospholipid classes have precluded direct quantitation ofstructurally diverse lipids by FAB-MS. Because ESI isrelatively insensitive to modest differences in surface activ-ities of phospholipid classes and subclasses, it has the ad-vantage of facilitating the simultaneous quantification oflipids having disparate surface activities. For example, thenegative-ion ESI mass spectrum of equimolar mixtures ofarachidonic acid (m/z 303), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA, m/z 674), POPE (m/z 717), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG, m/z 748),and POPS (m/z 761 and 380) (5 pmol of each was consumedduring a 1-min infusion at 1 A4/min of the mixture containing5 pmol/yl of each) in the presence of 30 pmol of NaOHcontains five singly-charged molecular ion peaks (i.e.,[M-H]I-), which are each within 5% of theoretical valuesafter correction for 13C isotope effects (Fig. 4A, horizontalbars) except for POPE. The intensity of the ethanolamineglycerophospholipid molecular ion was consistently less thanthose of the anionic phospholipid classes, a phenomenon

which results from the predominant zwitterionic character ofethanolamine glycerophospholipids. Ethanolamine glycero-phospholipids, in contrast to anionic phospholipids, must beinduced to lose a proton from their ammonium ion duringionization, and hence their quantitation requires a correctionfactor for direct comparisons to anionic phospholipids.Through examination of the molar ratio ofPOPG consumedto POPE consumed vs. the molecular ion peak intensity ratio(Fig. 4B), a correction factor of 1.50 + 0.07 for ethanolamineglycerophospholipids vs. anionic phospholipids (prior to cor-rection for the 13C isotope effect) was obtained from linearregression analysis (Fig. 4C). Similarly, alteration of themolar ratio of two lipids having widely disparate surfaceactivities [i.e., addition of various amounts of 1-palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) to asolution containing DPPC] also resulted in a linear correlationbetween their molar ratios and the ratio of their respectivesodiated molecular ion peak intensities in positive-ion ESImass spectra (slope = 1.00; r2 = 0.998). Finally, ESI-MS wasuseful for the quantitative analysis of choline glycerophos-pholipid subclasses. An equimolar mixture ofthe three majorcholine glycerophospholipid subclasses-plasmenylcholine,plasmanylcholine, and phosphatidylcholine [i.e., 1-O-(Z)-hexadec-1'-enyl-2-octadec-9'-enoyl-sn-glycero-3-phospho-choline (m/z 767), 1-O-hexadecyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine (m/z 769), and 1-hexadecanoyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine (m/z 783)]-resulted in three sodiated molecular ion peaks ([M+Na]+)which were each present in equal intensity (within 5%) (Fig.5).

In summary, these results demonstrate that after the simpleextraction of phospholipids from cellular sources, ESI-MSallows the direct structure determination and quantitation ofcritical phospholipid constituents from biologic samples. Theapplication of ESI-MS to the identification of individualphospholipid constituents comprising human erythrocyteplasma membranes resulted in the demonstration of the high

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100 769

80

Z%

0) 60

.> 40-

a:20-

740 750 760 770 780 790 800 810

m/z

FIG. 5. Positive-ion ESI mass spectrum of phospholipid sub-classes. Positive-ion ESI-MS of equimolar mixtures comprising thethree major choline glycerophospholipid subclasses (5 pmol/pl ofeach) demonstrated three molecular ion peaks-i.e., sodiated plas-menylcholine [1-O-(Z)-hexadec-1'-enyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine, m/z 767], sodiated plasmanyicholine (1-O-hexadecyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine, m/z769) and sodiated phosphatidylcholine (POPC, m/z 783)-withnearly identical peak intensities (within 5%).

content of plasmalogens, the selective enrichment of poly-unsaturated (four or more double bonds) fatty acids in theplasmenylethanolamine pool, and the predominance of poly-unsaturated fatty acids in serine glycerophospholipids. Col-lectively, these results demonstrate that ESI-MS of phos-pholipids is an enabling strategy for the direct structuredetermination and quantitative analysis of subpicomoleamounts of phospholipids derived from either biologicsources or synthetic processes. Accordingly, we anticipatethat ESI-MS will facilitate identification of the critical phos-pholipid pools undergoing accelerated catabolism duringcellular activation, thereby clarifying the contribution ofspecific intracellular phospholipases as mediators of signaltransduction in mammalian cells.

This research was supported by National Institutes of HealthGrants HL35864 and HL41250.

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