detection of individual phospholipids in lipid mixtures by matrix-assisted laser...

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Analytical Biochemistry 289, 202–216 (2001)doi:10.1006/abio.2000.4926, available online at http://www.idealibrary.com on

Detection of Individual Phospholipids in Lipid Mixtures byMatrix-Assisted Laser Desorption/Ionization Time-of-FlightMass Spectrometry: Phosphatidylcholine Preventsthe Detection of Further Species

Marijana Petkovic,1,2 Jurgen Schiller,2 Matthias Muller, Stefan Benard, Sabine Reichl,laus Arnold, and Jurgen Arnhold

Institute of Medical Physics and Biophysics, Medical Faculty, University of Leipzig, D-04103 Leipzig, Germany

Received July 21, 2000

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an estab-lished tool for the analysis of proteins, whereas itgained by far less interest in the field of lipid analysis.This method works well with phospholipids as well asorganic cell extracts and provides high sensitivity andreproducibility. The aim of the present paper is toextend our previous studies to the analysis of lyso-phospholipids and phospholipid mixtures. To studythe suitability of MALDI-TOF mass spectrometry forthe analysis of lysophospholipids, different phospho-lipids like phosphatidylcholine, phosphatidylethanol-amine, phosphatidylserine, phosphatidic acid, andphosphatidylinositol as well as their mixtures weredigested with phospholipase A2. Positive and negativeion mass spectra of all phospholipids before and afterdigestion were recorded. In all these cases, the molec-ular ions of the expected digestion products could bedetected and only a very small extent of further frag-mentation was observed. On the other hand, spectra ofphospholipid mixtures containing phosphatidylcho-line were strongly dominated by phosphatidylcholineand lysophosphatidylcholine signals, which preventedthe detection of further phospholipids even if thoselipids were present in comparable amounts. This is ofparamount interest for the analysis of tissue and cellextracts. © 2001 Academic Press

Key Words: MALDI-TOF MS; phospholipase A2; phos-pholipids; lysophospholipids.

1 To whom correspondence should be addressed at Institute ofMedical Physics and Biophysics, University of Leipzig, Liebigstrasse27, D-04103 Leipzig, Germany. Fax: 149 341 97 15 709. E-mail:
[email protected].
2 Both authors contributed equally to this work.

202

Phospholipids are main constituents of biologicalmembranes, providing an important source for lipid-derived second messengers. Lipid-derived second mes-sengers like inositolphosphates, diacylglycerols, andphosphatidic acids are implicated in the regulation ofmany cellular functions including proliferation, differ-entiation, and a number of specific functions (1–3).Phospholipase A2 acts specifically at the sn-2 positionof a phospholipid (4) producing a free and mainly un-saturated fatty acid as well as the corresponding lyso-phospholipid. Lysolipids were recently discovered asnovel intra- and intercellular signaling molecules,involved in the activation of many specific enzymes(5–10).

The investigation of the activity of various phospho-lipases usually requires radioactive labeling, e.g., with3H or 32P and derivatization of phospholipids for fur-ther separation and detection by TLC3 or HPLC (11–14). In some particular cases, it is also possible tocharacterize the reaction products of phospholipases(e.g., choline or phosphorylcholine) by their known mo-lecular weights (15). All available methods are usuallyindirect and time-consuming. Unfortunately, there arenot yet appropriate methods for the routine determi-nation of lysophospholipids in the cells or in the corre-sponding lipid extracts.

3 Abbreviations used: DHB, 2,5-dihydroxybenzoic acid; ESI, elec-trospray ionization; FAB, fast atom bombardment; HPLC, high-performance liquid chromatography; MALDI-TOF MS, matrix-as-sisted laser desorption/ionization time-of-flight mass spectrometry;LPL, lysophospholipid; MS, mass spectrometry; PA, phosphatidicacid; PBS, phosphate-buffered saline; PE, phosphatidylethanol-amine; PI, phosphatidylinositol; PLA2, phospholipase A2; PC, phos-
phatidylcholine; PS, phosphatidylserine; PL, phospholipids; TFA,trifluoroacetic acid; TLC, thin-layer chromatography.
0003-2697/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

203MASS SPECTROMETRY OF PHOSPHOLIPID MIXTURES

If mass spectrometry is used that is usually done byelectron impact ionization in that way that the sampleis saponified under alkaline conditions and the result-ing free fatty acids (mainly after derivatization withtrimethyl-chlorsilane) are determined. Unfortunately,this tedious procedure does not provide any informa-tion on the actual lipid composition of the sample, i.e.,in what lipid those fatty acids have been located pre-viously. This serious drawback of this most commonionization method of mass spectrometry has stimu-lated the development of more sophisticated methodsas plasma desorption (PD), fast atom bombardment(FAB), and as electrospray ionization (ESI). All thesetechniques are in principle useful for lipid analysis andthere is still no agreement what method works best.FAB has been used in most cases but is more and morereplaced by ESI (16).

Matrix-assisted laser desorption and ionization(MALDI-TOF) mass spectrometry is an establishedtool for the analysis of proteins, carbohydrates, andnucleic acids (17–19). Although it was shown thatMALDI methodology has a great potential for lipidanalysis (20), this method gained only less interest inthis field. However, recently it has been shown thatdifferent classes of phospholipids, as well as diacyl-glycerols, can be easily and accurately analyzed byMALDI-TOF MS and that 2,5-dihydrobenzoic acid(DHB) is the matrix of choice (21–23). An importantadvantage of MALDI-TOF MS in lipid analysis is thatboth—the analyte and the matrix—are easily solublein organic solvents. Therefore, homogeneity of the ma-trix/analyte mixture, which is most important for therecording of reproducible spectra, is excellent and thesignal to noise ratio of the spectra is superior to otherionization techniques (21). A further advantage ofMALDI-TOF is that mainly singly charged molecularions are formed (24). This leads to mass spectra thatare easy to analyze, since each individual moleculeresults in a single peak. Besides the proton adductsNa1 and K1 adducts are detectable, but their formationcan be minimized by experimental conditions. There-fore, mainly protonated molecular ions [M 1 1]1 areformed and no major peak overlap occurs. Additionally,it has been clearly shown that the application of suit-able internal standards allows an at least semiquanti-tative determination of lipids by MALDI-TOF MS in avery fast and convenient way (23).

Since under natural conditions mixtures of differentlipids with strongly varying fatty acid composition oc-cur, our intention was to analyze the suitability ofMALDI-TOF mass spectrometry for the analysis ofmixtures of different phospholipids and lysophospho-lipids. We will show that MALDI-TOF mass spectrom-etry exhibits different sensitivity toward individualclasses of phospholipids in a mixture, with phosphati-
dylcholine being the most easily detectable of all. We
shall also try to support the standpoint that themethod represents a powerful tool in the field of lipidresearch.

MATERIALS AND METHODS

Materials

All lipids used in this study were purchased fromAvanti Polar Lipids Inc. (Alabaster, AL). All phos-pholipids were obtained as 20 mg/ml solutions inchloroform. The following fatty acid compositionswere provided by the manufacturer: egg yolk phos-phatidylcholine—16:0 (34%), 16:1 (1%), 18:0 (10%),18:1 (31%), 18:2 (17%), and 20:4 (3%); brain phospha-tidylserine—18:0 (41%), 18:1 (31%), and 22:6(8.45%); egg phosphatidylethanolamine—16:0 (16%),18:0 (24%), 18:2 (14%), and 20:4 (16%); liver phos-phatidylinositol—18:0 (41%), 18:1 (17%), 18:2 (6%),20:3 (10%), and 20:4 (17%); and egg phosphatidicacid—16:0 (34%), 16:1 (1%), 18:0 (10%), 18:1 (31%),18:2 (17%), and 20:4 (3%). For some comparativemeasurements synthetic dipalmitoyl phosphatidicacid from Fluka (Neu Ulm, Germany) was used. Hogpancreas phospholipase A2 was purchased fromFluka (Neu Ulm, Germany), showing an activity of561 U/mg protein.

Matrix for mass spectrometry, namely, 2,5-DHB,chloroform, and methanol (with less than 0.00005% ofsodium and potassium) were purchased from Fluka(Germany). All chemicals were of highest commerciallyavailable purity and were used without any furtherpurification.

Sample and Matrix Preparation

For all samples, a 0.5 M 2,5-DHB solution in meth-anol containing 0.1% trifluoroacetic acid (TFA) wasused. TFA was used because small amounts of TFAenhance the signal to noise ratio of lipid spectra (21).No degradation of the phospholipids was observed inthe presence of TFA at the concentration used in ourexperiments.

Individual lipids were diluted with chloroform to 1mg/ml and were directly transferred to the sampleplate as 1-ml droplets and the same volume of thematrix solution was applied afterward. While phospho-lipid mixtures were analyzed, the total lipid concentra-tion was between 0.75 and 1.5 mg/ml and the concen-tration of the individual phospholipid within themixture was 0.5 mg/ml. In experiments designed toinvestigate the influence of PC on the detectability offurther phospholipids in the mixtures the concentra-tion of PC was varied from 0.05 to 0.5 mg/ml, whereasthe concentration of other phospholipids present in themixtures was kept constant (0.5 mg/ml). For all
MALDI-TOF MS measurements, the same volume

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204 PETKOVIC ET AL.

(1 ml) of different phospholipid mixtures was applied tothe sample plate followed by the addition of the samevolume of matrix. Samples were rapidly dried under awarm stream of air in order to remove the organicsolvent as fast as possible. This procedure gave the byfar best crystallization properties and led to a consid-erable increase in reproducibility of the spectra.

Lipid Digestion with Hog Pancreas Phospholipase A2

Solutions of individual lipids or phospholipid mix-tures with a final total lipid concentration of 0.75 up to1.5 mg/ml were evaporated to dryness and resus-pended in 100 mM PBS, pH 7.4, and incubated withhog pancreas phospholipase A2 (561 U/ml) for 4 h at37°C. These digestion conditions were chosen as theygave complete digestion of most of the phospholipidsused. As control, lipids were incubated without theenzyme under the same experimental conditions. Di-gestion was terminated by the addition of an ice-coldmixture of chloroform/methanol/0.1 M HCl (1/2/0.03,v/v/v). The chloroform phase was separated by cen-trifugation (800g) and directly used for mass spectrom-etry.

Mass Spectrometry

All MALDI-TOF spectra were acquired on a VoyagerBiospectrometry workstation (PerSeptive Biosystems,Framingham, MA). The system utilizes a pulsed nitro-gen laser, emitting at 337 nm. Pressure in the ionchamber was maintained between 1 3 1027 and 4 31027 Torr. A two-stage acceleration device allows toapply delayed extraction conditions (DE mode), im-proving both mass resolution and mass accuracy (24).The formed ions were accelerated to a kinetic energy of20 keV within the ion source. In order to enhance thespectral resolution, the device was used in the reflectormode, so that the total field-free time-of-flight distancewas 2 m. An internal calibration was performed bysetting the peak of the protonated DHB matrix to itsappropriate value (155.034 Da). This procedure wasfound to be sufficiently accurate for the determinationof the individual molecular masses.

To enhance the reproducibility, 128 single shots fromthe laser were averaged for each mass spectrum. Thelaser power was kept about 10% over threshold toobtain the best signal to noise ratio. All lipid spectrawere acquired using a low-mass gate at 400 Da toprevent the detector from saturation by ions arisingfrom matrix degradation or photochemical reactions.

RESULTS AND DISCUSSION

Since phospholipids of biological origin, e.g., cell ortissue extracts, consist of a mixture of different classes
of phospholipids with a great variation in fatty acid 5
chain composition, we have designed experiments toinvestigate the suitability of MALDI-TOF mass spec-trometry for the detection of certain lipids in crudemixtures. First, we have addressed the questionwhether DHB (that is a recommended matrix for theanalysis of phospholipids due to its low yield of frag-mentation products) (21, 22) may influence the detec-tion of phospholipids. Next, we analyzed the spectra ofpure phospholipids of biological relevance, and finallywe analyzed mixtures of phospholipids in order to in-vestigate the influence of a particular phospholipidclass on the shape of the spectra.

For all spectra, in the positive as well as in thenegative ion mode, we have used a 0.5 M DHB solutionin methanol as matrix. The matrix by itself, however,yields a number of peaks, especially in the negative ionmode, and at lower m/z ratios in the positive massspectra. Additionally, the presence of inorganic salts inthe solvents used, e.g., Na1 or K1, leads to the forma-ion of further matrix adducts. Therefore, it was ourery first aim to show that the presence of the DHBatrix does actually not complicate the interpretation

f mass spectra of lipids and that matrix peaks can beasily distinguished from peaks arising from phospho-ipids.

In Fig. 1 different laser desorption–TOF mass spec-ra of the applied DHB matrix are shown: Figs. 1a andb were recorded in the positive ion mode, whereas inigs. 1c and 1d the corresponding negative ions wereetected; Figs. 1a and 1c represent DHB dissolved inure methanol, whereas the solvent for Figs. 1b and 1das saturated with sodium chloride. The assignment ofll major peaks is given in Fig. 1. The neutral form (M)f DHB has a molecular weight of 154 Da. Therefore, itroduces an intense signal at m/z 5 153 [M 2 H]2 in

the negative ion mode. The corresponding signal in thepositive mode is found at m/z 5 155 [M 1 H]1 sinceone additional proton is needed for the conversion ofthe neutral molecule to a positively charged molecularion. The peaks at m/z 5 177 (D 5 22 Da) and m/z 5

99 (D 5 44 Da) in the positive mass spectra corre-pond to molecular ions containing one or two sodiumtoms, respectively. The peak at m/z 5 137 can bexplained by water elimination subsequent to protona-ion. Such a fragmentation is typical for moleculesontaining hydroxyl groups (25, 26). In contrast to thepectra of DHB in pure methanol the presence of NaClbviously leads to an enhancement of the formation ofssociation products of DHB with a higher moleculareight. This behavior is stronger expressed in the neg-tive ion mode (cf. Figs. 1c and 1d).Since we are primarily interested in phospholipidsith masses higher than 400 Da, all spectra of lipidsere recorded with a low “mass gate” at m/z 5 400 tovoid most of matrix peaks. Only the matrix peaks at
51 (in some cases also at 567 for [3M 2 3H 1 3Na 1

epacon


3K]1 and 727 [4M 2 4H 1 5Na]1) can influence thedetection of phospholipids in the positive ion mode.However, the spectra in the negative ion mode aremore influenced by the presence of matrix peaks. Thelower sensitivity of the negative ion mode seems toresult from the enhanced number of matrix peaks (dis-cussed later in more detail).

Positive Ion Mass Spectrometry of Phospholipids

Our experiments are focused on PC, PS, and PEbecause they are major constituents of cell membranes.The chemical structures of phospholipids analyzed inthis paper, i.e., PC, PE, PS, PA, and PI are given in Fig.2. The structure of lysophosphatidylcholine is given asan example for the structure of lysophospholipids con-taining a free hydroxyl group in the sn-2 position.Spectra of the corresponding phospholipids are shownin Fig. 3, and all peaks identified are listed in Table 1.All these lipids are easily detectable by MALDI-TOF

FIG. 1. Laser desorption–TOF mass spectra of pure DHB (2,5-dihydM solution in methanol containing 0.1% TFA: (a and b) Positive ioncontained no additional sodium chloride, whereas (b) and (d) were prall spectra were recorded using a reflectron and delayed extraction

mass spectrometry, however, with different sensitivity.

The positive ion mass spectrum of egg yolk PC (Fig.3a) consists of several isotopically resolved peaks (seeinset in Fig. 3a). The most intense peaks of protonadducts correspond to palmitoyl-linoleoyl-PC (758.6,[M 1 H]1), palmitoyl-oleoyl-PC (760.6, [M 1 H]1),palmitoleoyl-arachidonoyl-PC (780.6, [M 1 H]1),palmitoyl-arachidonoyl-PC (782.6, [M 1 H]1), andstearoyl-arachidonoyl-PC (810.6, [M 1 H]1) (cf. Table1). The peaks at 780.6 and 782.6 can be also assigned to18:2/18:3 PC or 18:1/18:3 PC, respectively, althoughthis assignment is not of high probability. The sodiumadducts of palmitoyl-linoleoyl-PC and palmitoyl-oleoyl-PC also contribute to the peaks at 780.6 and782.6, respectively, since in the absence of sodium (i.e.,after washing the sample with distilled water) the in-tensity of those peaks decreases (data not shown).Fragmentation products of PC that may be due to theloss of the choline head group (21) are only detectableto a minor extent under our experimental conditions,

ybenzoic acid), which was used as matrix in all experiments as a 0.5de spectra and (c and d) negative ion spectra. Samples in (a) and (c)red with sodium chloride-saturated methanol. For better resolutionditions.

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implying the suitability of this method also for mixture

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206 PETKOVIC ET AL.

analysis. The characteristic peaks at 551 and 567 (la-beled with an asterisk, Fig. 3) that are detected in allpositive ion spectra correspond to the DHB matrixproduct (cf. Fig. 1).

The corresponding positive ion mass spectrum of eggPE is shown in Fig. 3b. PE—like PC—yields molecularions that arise from the addition of one hydrogen or onesodium ion. Egg PE consists of a large variety of dif-ferent species. The identity of peaks is given in Table 1.The most intense peaks correspond to PEs with thefollowing fatty acid composition: 16:0/20:4 (740.6 and762.6), 18:0/18:2 (744.6, 766.6, and 788.6), and 18:0/20:4 (768.6, 790.6, and 812.6). The spectrum in Fig. 3bcontains also a number of peaks between 590 and 650,corresponding to fragmentation products of PE thatare probably generated by a breakdown of the unsat-urated fatty acid residue.

The PS yields peaks at higher m/z ratios than PCand PE (Fig. 3c; Table 1). Since PS represent acidiclipids (cf. Fig. 2), two positive charges, i.e., two positiveions, are needed to produce a positively charged molec-ular ion. Typically for charged analytes, different ra-tios of H1, Na1, and K1 result in a more complex peakpattern of the PS spectra than in the case of PC andPE. The most abundant PS species present in the sam-ple contains stearic and oleic acid. The correspondingmolecular ions for PS (18:0, 18:1) are detectable at812.6 ([M 1 H 1 Na]1), 828.6 ([M 1 H 1 K]1), 834.6[M 1 2Na]1), and 850.6 ([M 1 Na 1 K]1). Additional

peaks at higher molecular weight correspond to PS

FIG. 2. Structures of phospholipids investigated in this study. Thephosphatidylserine (PS), phosphatidic acid (PA), and phosphatidylinline as an example for lysophospholipids. R, a fatty acid residue; posilocated under physiological conditions, i.e., phosphate groups (negcharges).

species containing docosahexaenoic acid (cf. Table 1).

The peaks at 551 and 567 arise from matrix adducts(Fig. 3c, labeled with an asterisk).

Additional peaks were found at 605.5 and betweenabout 740 and 760, corresponding to fragmentationproducts of different PS species. The chemical struc-ture of these fragmentation products remains yet un-known. Their intensities, however, decreased when thelaser power was lowered (data not shown). A similarobservation has been made for the peaks between 590and 650 of the PE samples.

Positive ion MALDI-TOF mass spectra of two acidiclipids, PA and PI, are shown in Figs. 3d and 3e, respec-tively. PAs are phospholipid species that bear two neg-ative charges (cf. Fig. 2) and, therefore, need compen-sation with three cations to be detectable as positiveions. Two intense peaks at 697.5 and 719.5 contributeto PA (16:0, 20:4) upon addition of three protons or onesodium and two protons, respectively. This assignmentwas additionally confirmed by the addition of highconcentrations of NaCl that led to the replacement ofprotons by sodium, i.e., an enhancement of the sodiatedmolecular ions (data not shown). Additionally,stearoyl-linoleoyl-PA yields a small peak at m/z 5745.5 (cf. Table 1). All other peaks are indicated inTable 1. To a lesser extent some yet unidentified frag-mentation products of PA with a molecular weightlower than 400 were observed when the measurementswere carried out without the “low mass gate” (data notshown).

The positive ion mass spectrum of PI is shown in Fig.

mulae of phosphatidylcholine (PC), phosphatidylethanolamine (PE),ol (PI) are presented as well as the formula of lysophosphatidylcho-and negative charges are indicated on the positions where they are

ve charges), and choline and ethanolamine head groups (positive

forosittive

3e. The different proton and sodium adducts (see Table


1) of the following PI species are clearly detectable: PI(18:0, 18:2), PI (18:0, 18:1), PI (18:0, 20:4), and PI (18:0,20:3) (Fig. 3e; Table 1). Nonlabeled peaks at m/z 623.3and 645.3 correspond to lyso-PI, which might be acontaminant of the commercial lipid preparation.

Obviously, the peak intensity in the positive ionmass spectra decreases with increasing polarity ofphospholipid. The peak intensity, however, does notreflect the concentration of different individual phos-pholipid species (cf. Fig. 3) since all phospholipids were

FIG. 3. Positive ion MALDI-TOF mass spectra of egg yolk PC (a), eg(m/z 5 750–800) of the PC spectrum to show that peaks are isotopicarising from matrix (DHB) adducts are labeled with an asterisk.

applied in nearly equal amounts and with similar fatty

acid distribution. Since peak intensity, e.g., for PC ismuch higher than that for PS, or PI, the polarity andthe charge state must play an important role. Thislower sensitivity of MALDI-TOF toward the detectionof phospholipids of higher polarity could make thequantification of individual phospholipids in a phos-pholipid mixture difficult. Additionally, the molecularweight of lipids has an influence on the sensitivity.Asbury et al. (27) found that peak intensity of triacyl-glycerides correlates reciprocally with the number of

E (b), brain PS (c), PA (d), and liver PI (e). (Inset) An expanded regiony resolved. Peaks are labeled according to their m/z ratios and peaks

g Pall

carbons.

208 PETKOVIC ET AL.

Negative Ion Mass Spectrometry of Phospholipids

The phospholipids PS, PA, and PI produce nega-tively charged molecular ions. The negative ion

TABLE 1

Assignment of Peaks Detectable in the Positive Ion MassSpectra Sorted by the m/z ratio

Peak position(m/z) Peak assignment

;590–650 Fragmentation products of PE;740–760 Fragmentation products of PS

551 Matrix adduct567 Matrix adduct

605.5 Fragmentation product of PS695.5 [PA(16:1,20:4)13H]1 or [PA(16:0,20:3)13H]1

697.5 [PA(16:0,20:4)13H]1

717.5 [PA(16:1,20:4)12H1Na]1 or[PA(16:0,20:3)12H1Na]1

718.6 [PE(16:0,18:1)1H]1 or [PE(16:1,18:0)1H]1

719.5 [PA(16:0,20:4)12H1Na]1

735.5 [PA(16:0,20:4)12H1K]1

738.6 [PE(16:1,20:4)1H]1

740.6 [PE(16:0,20:4)1H]1

741.5 [PA(16:0,20:4)1H12Na]1

744.6 [PE(18:0,18:2)1H]1

745.5 [PA(18:0,18:2)1H12Na]1

746.6 [PE(18:0,18:1)1H]1

757.5 [PA(16:0,20:4)1H1Na1K]1

758.6 [PC(16:0,18:2)1H]1

760.6 [PC(16:0,18:1)1H]1

760.6 [PE(16:1,20:4)1Na]1

762.6 [PE(16:0,20:4)1Na]1

766.6 [PE(18:0,18:2)1Na]1

768.6 [PE(18:0,20:4)1H]1 or [PE(18:0,18:1)1Na]1

780.6 [PC(16:1,20:4)1H]1 and [PC(16:0,18:2)1Na]1

782.6 [PC(16:0,20:4)1H]1 and [PC(16:0,18:1)1Na]1

788.6 [PC(18:0,18:1)1H]1

788.6 [PE(18:0,18:2)2H12Na]1 or [PE(18:1,20:4)1Na]1

790.6 [PE(18:0,20:4)1Na]1

808.6 [PC(18:1,20:4)1H]1

810.6 [PC(18:0,20:4)1H]1

812.6 [PE(18:0,20:4)2H12Na]1

812.6 [PS(18:0,18:1)1H1Na]1

828.6 [PS(16:0,20:4)12Na]1 or [PS(18:0,18:1)1H1K]1

834.6 [PS(18:0,18:1)12Na]1

850.6 [PS(16:0,20:4)2H13Na]1 or[PS(18:0,20:4)1H1K]1 or[PS(18:0,18:1)1Na1K]1

856.6 [PS(18:0,18:1)2H13Na]1

858.6 [PS(18:0,22:6)1H1Na]1

880.6 [PS(18:0,22:6)12Na]1

885.6 [PI(18:0,18:2)1H1Na]1

887.6 [PI(18:0,18:1)1H1Na]1

907.6 [PI(18:0,18:2)12Na]1

909.6 [PI(18:0,18:1)12Na]1

911.6 [PI(18:0,20:3)1H1Na]1

931.6 [PI(18:0,20:4)12Na]1

933.6 [PI(18:0,20:3)12Na]1

Note. The table comprises the isolated lipids as well as the phos-pholipid mixtures.

MALDI-TOF mass spectra of PS, PA, and PI are given

in Fig. 4. The phosphatidylinositols used in our exper-iments yield four peaks in the negative ion mode at861.6 (18:0/18:2), 863.6 (18:0/18:1), 885.6 (18:0/20:4),and at 887.6 (18:0/20:3) (Fig. 4a; Table 2). This is inaccordance with the fatty acid composition of PI pro-vided by the manufacturer, as well as with the positiveion mass spectra of PI (Fig. 3e). The number of peaksarising from phospholipids in the negative ion mode isconsiderably lower than in the positive ion mode. Thediversity of spectra is lower because less positive ions(e.g., hydrogen, sodium or potassium) are required forcharge compensation. Molecular ions are generated bythe abstraction of one proton, i.e., mainly [M 2 H]2

ions are present and, therefore, the molecular weight ofphospholipids is shifted by 2 Da toward lower molecu-lar weights in comparison to the corresponding posi-tively charged form (if charge compensation only byprotons is assumed). One additional peak in the spec-trum of PI of much lower intensity is detected at 599.3and can be assigned to lyso-PI (Fig. 4a; Table 2). Theintensity of this peak strongly increases after digestionwith phospholipase A2 (cf. below) what further con-firms the peak identity.

Figure 4b shows the negative ion mass spectrum ofPA. Phosphatidic acid bears two negative charges and,thus, the addition of one cation is necessary for thedetection of PA as a singly charged molecular ion.Unfortunately, even when applied in higher concentra-tions (up to 3 mg/ml), egg PA was not detectable in thenegative ion mass spectra under our experimental con-ditions. Since PA used in our study actually consists ofseveral species, we assume that the concentrations ofeach individual species are below the detection limit,i.e., yield of individual negative ions is too low for ourdetection system. On the other hand, we were able todetect a synthetic PA (16:0,16:0) in the negative ionmode (data not shown).

The negative ion mass spectrum of PS is given in Fig.4c. As in the case of PI, no additional cations arerequired for charge compensation of PS. Stearoyl-oleoyl-PS yields one peak at 788.6, whereas the PScontaining docosahexaenoic acid (22:6) yields one peakat 834.6. The peaks at 820.6 and 705.6 could not beidentified.

In all spectra presented in Fig. 4, the overall signalheight is somewhat lower than in the positive ion mode(Fig. 3). The number of peaks that arise from matrix(peaks labeled with an asterisk) is also higher andmight contribute to the lower sensitivity of this modetoward the detection of phospholipids. However, due tosimplicity and higher homogeneity toward distributionof adducts, negative ion mode spectra are often helpfulfor the final assignment of molecular species detected
as positive ions.

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Mass Spectrometry of Lysophospholipids

Lysophospholipids represent naturally occurringphospholipid species present in biological membranesin rather low concentrations (11). In order to investi-gate the suitability of MALDI-TOF mass spectrometryfor the analysis of lysophospholipids as well as mix-tures of different lysophospholipid species, we sub-jected all investigated phospholipids to a digestionwith hog pancreas phospholipase A2. It is known that

hospholipase A2 hydrolyses the ester bond of phospho-lipids in the sn-2 position (4). Since naturally occurring

hospholipids contain mainly a saturated or monoun-

FIG. 4. Negative ion mass spectra of PI (a), PA (b), and PS (c). Pefrom matrix adducts are labeled with an asterisk.

TABLE 2

Assignment of Peaks Detected in the Negative Ion MassSpectra of Pure PI and PS

Peak position (m/z) Peak assignment

599.3 [Lyso-PI(18:0)2H]2

705.6 Not identified788.6 [PS(18:0,18:1)2H]2

820.6 Not identified834.5 [PS(18:0,22:6)2H]2

861.6 [PI(18:0,18:2)2H]2

863.6 [PI(18:0,18:1)2H]2

885.6 [PI(18:0,20:4)2H]2

t887.6 [PI(18:0,20:3)2H]2

aturated fatty acid at the sn-1 position phospholipase2 digestion of phospholipids yields as main products

lysophospholipids with the corresponding saturated ormonounsaturated fatty acid residue.

As expected, in the spectra of PC digested with phos-pholipase A2 (Fig. 5a; Table 3) new peaks at lower m/zratios arise besides the peaks from undigested PC.Three different lyso-PCs are detected: The hydrogenand the sodium adducts of 1-palmitoyl-glycero-phos-phorylcholine give peaks at m/z 5 496.3 and m/z 5518.3, respectively, whereas the peak at 522.3 corre-sponds to the protonated form of 1-oleoyl-glycero-phos-phorylcholine. Finally, the peaks at 524.4 and at 546.3correspond to the protonated and sodiated forms of1-stearoyl-glycero-PC, respectively. The arising of onlythree digestion products is in agreement with theknown cleavage sites of phospholipase A2 and exclu-sively lyso-PCs with a residual saturated, palmitic orstearic, or monounsaturated fatty acid, i.e., oleic acid,are detectable after digestion with phospholipase A2.

Like in the case of PC, PE digested with phospho-ipase A2 yields molecular ions corresponding to the

proton and the sodium adducts of lyso-PE (16:0) (454.3,476.3, 498.3) and (18:0) (482.3, 504.3, 526.3) (Fig. 5b;Table 3). Moreover, the phospholipase A2 digestion ofPE and the cleavage of the fatty acid at the sn-2 posi-

are labeled according to their molecular masses and peaks arising

ion decreases the intensities of fragmentation prod-

) ly

210 PETKOVIC ET AL.

ucts detectable in the spectra of pure PE (Fig. 3b) atleast below the detection limit of our system. Thisindicates once more that less intense peaks detected inthe spectra of pure PE in the range of about 590 up to640 arise from the fragmentation of PE. Astonishingly,in contrast to PC (Fig. 5a) there was no residual (un-digested) PE species detectable. The concentration ofthe residual PE might be below the detection limit ofour system. This also might be explained by a higheractivity of phospholipase A2 toward the digestion of PEthan PC, at least with the fatty acid composition thatwas used in this study and under our experimentalconditions.

In accordance with the spectrum of pure PS, lyso-PSproduced by the digestion of PS with PLA2 give mainlythe expected positive molecular ions generated by theaddition of a mixture of different cations (Fig. 5c; Table3). Lyso-PS (18:0) containing one proton at the phos-

FIG. 5. Positive ion mass spectra of lyso-phospholipids: (a

phate residue, as well as at the carboxylate group (cf.

Fig. 2), possesses a molecular weight of 525.3 and givestherefore a peak at 526.3 upon cationization by oneadditional proton. Since these three protons can, how-ever, also be replaced by sodium ions, one can alsodetect peaks at 548.3, 570.3, and finally 592.3, whereall protons are exchanged by sodium. In agreementwith the positive ion mass spectra, the negative ionmass spectra (data not shown) of digestion products ofPS yield only a single peak arising from the negativeion of lyso-PS (18:0). This product is generated uponloss of one proton and gives a peak at 524.3 (Table 3)that additionally confirms the identity of the peaksdetected in the positive ion mass spectra (Fig. 5c). Likein the case of PE, there is no undigested PS detectable.

After digestion of PA with PLA2, the expectedlyso-PA (16:0) was generated giving molecular ionswith different ratios between H1 and Na1 (Fig. 5d,Table 3). Since the molecular weight of the completely

so-PC; (b) lyso-PE; (c) lyso-PS; (d) lyso-PA; and (e) lyso-PI.

dissociated form of that lyso-PA is 408, the overcom-

N

wah

(l


pensation of the two negative charges leads to a mo-lecular ion at 433.2 ([M 1 2H 1 Na]1). The exchange ofthe protons by sodium results in the peaks at 455.2 and477.2. The identity of the two latter peaks was addi-tionally confirmed by applying higher sodium concen-trations. In this case, the intensity of both peaks, at455.2 and 477.2, considerably increases (data notshown). The spectra show also two peaks at 461.2 and483.2 for lyso-PA (18:0) corresponding to [M 1 2H 1

a]1 and [M 1 H 1 2Na]1, respectively. Like PA,lyso-PA is a highly acidic phospholipid, but no peakscorresponding to this molecular species were detect-able in the negative ion mode under our experimentalconditions (data not shown).

On the other hand, we were able to detect the lyso-PIgenerated after digestion of PI with phospholipase A2

(cf. Table 3). The expected lyso-PI yields one peak at599.3 in the negative ion mass spectra that corre-sponds to the completely dissociated form of lyso-PI(18:0). In the positive ion mass spectra, the samelyso-PI yields two peaks that are formed by the addi-tion of one proton and one sodium (623.3) or two so-dium ions (645.3) (Fig. 5e; Table 3). This indicates oncemore that heterogeneity of the starting material isstrongly reduced by the phospholipase A2 digestion

hereby the unsaturated fatty acid was split. Remark-bly, the peak intensity of the formed lyso-PI is much

TABLE 3

Assignment of Peaks Detected in the Positive as Well asthe Negative Ion Mass Spectra of Digestion Products of Phos-pholipids with Phospholipase A2

Peak position (m/z) Peak assignment

433.2 [Lyso-PA(16:0)12H1Na]1

454.3 [Lyso-PE(16:0)1H]1

455.2 [Lyso-PA(16:0)1H12Na]1

461.3 [Lyso-PA(18:0)12H1Na]1

476.3 [Lyso-PE(16:0)1Na]1

477.2 [Lyso-PA(16:0)13Na]1

482.3 [Lyso-PE(18:0)1H]1

483.3 [Lyso-PA(18:0)1H12Na]1

496.3 [Lyso-PC(16:0)1H]1

498.3 [Lyso-PE(16:0)2H12Na]1

504.3 [Lyso-PE(18:0)1Na]1

518.3 [Lyso-PC(16:0)1Na]1

522.3 [Lyso-PC(18:1)1H]1

524.3 [Lyso-PS(18:0)2H]2

524.4 [Lyso-PC(18:0)1H]1

526.3 [Lyso-PS(18:0)12H]1

526.3 [Lyso-PE(18:0)2H12Na]1

546.3 [Lyso-PC(18:0)1Na]1

548.3 [Lyso-PS(18:0)1H1Na]1

570.3 [Lyso-PS(18:0)12Na]1

592.3 [Lyso-PS(18:0)2H13Na]1

599.3 [Lyso-PI(18:0)2H]2

623.3 [Lyso-PI(18:0)1H1Na]1

645.3 [Lyso-PI(18:0)12Na]1

igher than that of pure PI (cf. Fig. 3e). However, this

does not correspond to a higher concentration of lyso-PIbut to the higher sensitivity of MALDI-TOF mass spec-trometry towards phospholipids of lower molecularmasses (see discussion above).

Since all MALDI-TOF measurements were carriedout with the “low mass gate” at 400, the free fatty acidsreleased by digestion of phospholipids with phospho-lipase A2 were not detectable.

Finally, we also tried to digest naturally occurringsphingomyeline with phospholipase A2. However, inthat case there was no difference between the spectrabefore and subsequent to phospholipase A2 digestiondata not shown). This clearly indicates that phospho-ipase A2 used in our study does not work on sphingo-

myeline. This experiment also shows that phospho-lipase A2 itself does not confer any additional peaks.

Mass Spectrometry of Phospholipid Mixtures

Equal amounts of different phospholipids weremixed and analyzed by MALDI-TOF mass spectrome-try. As a representative example, the positive ion massspectra of mixtures of different phospholipids are givenin Fig. 6. Phosphatidic acid and PS are present in allsamples. The spectral regions where these phospholip-ids are detectable are emphasized by a gray bar. Phos-phatidylcholine (Fig. 6a), PE (Fig. 6b), or PI (Fig. 6c)was mixed with PA and PS. The following mixtureswere also tested: PE/PS/PI, PA/PC/PI, PC/PS/PI, PE/PC/PS, PA/PC, PC/PI, and PS/PC (data not shown), ata total lipid concentration of 1.5 mg/ml for the mixtureof three phospholipids and 1 mg/ml for the mixture oftwo phospholipids. The concentration of each phospho-

FIG. 6. Typical positive ion mass spectra of mixtures of threedifferent phospholipids: (a) PA/PC/PS; (b) PA/PE/PS; and (c) PA/PS/PI. The spectral region where PA and PS are detectable are empha-sized by a gray bar. The total lipid concentration was 1.5 mg/ml, andthe final concentration of an individual phospholipid in the mixture
was 0.5 mg/ml. All spectra were recorded with 2,5-DHB as matrixand using a reflectron as well as delayed extraction conditions.

212 PETKOVIC ET AL.

lipid in the corresponding mixture was 0.5 mg/ml. Themost intriguing outcome of these experiments is that inthe presence of PC peaks arising from further speciesare suppressed even if they are present in amountscomparable to PC (an example is shown in Fig. 6a). PCdominates all spectra irrespectively on the nature offurther phospholipids in the mixture. Since PC is aneutral phospholipid and it is known that such phos-pholipids are more sensitively detectable as positiveions than acidic ones, we wanted to check whether thesecond neutral phospholipid, PE, has the same influ-ence on the detectability of PS, PA, or PI. The corre-sponding positive ion mass spectrum of a PA/PE/PSmixture is shown in Fig. 6b, and it is clearly evidentthat peaks arising from PA and PS are not suppressedby the presence of PE. Therefore, we assume that PE,in contrast to PC, does not decrease the detectability ofother phospholipids. This was also confirmed when amore complex mixture of phospholipids was analyzedby MALDI-TOF (data not shown). Finally, when allacidic phospholipids, i.e., PA, PS, and PI, are present inthe mixture, all of them are detectable with roughlythe same sensitivity (Fig. 6c).

Most probably, the preformed positive charge of PC(due to the presence of the quaternary ammonia group)enhances the detectability of PC in comparison to otherphospholipids and, therefore, PC has the lowest detec-tion limit by positive ion MALDI-TOF mass spectrom-etry. Accordingly, Schiller et al. (28) were able to detectexclusively PC and sphingomyeline—which also pos-sesses a quaternary ammonia group—in the organicextracts of human spermatozoa by MALDI-TOF massspectrometry. Therefore, the presence of PC in crudeextracts of biological material may cause problems forthe detection of different phospholipid classes, sincePC is highly abundant, e.g., in the membranes of poly-morphonuclear leukocytes (11).

Spectra of phospholipid mixtures were also recordedin the negative ion mode, but in this case, only someweak peaks of PS and PI were detectable (data notshown) confirming our previous statements on the sen-sitivity of the negative ion mode.

Subsequently to the analysis of phospholipid mix-tures, where we have shown that the presence of PCdecreases the detectability of other phospholipids, thequestion of the detectability of different lysophospho-lipid classes in the presence of lyso-PC arises. To checkthis, we have subjected all phospholipid mixtures pre-viously analyzed by MALDI-TOF mass spectrometry todigestion with phospholipase A2.

A typical set of data is presented in Fig. 7. Here, thepositive ion MALDI-TOF mass spectra of the digestionproducts of PA/PC/PS (Fig. 7a), PA/PE/PS (Fig. 7b),and PA/PS/PI (Fig. 7c) are given. Peaks are labeledaccording to their molecular masses, and peak identity
is given in Table 3. Like in the case of the pure phos-
pholipid mixtures, all spectra could be easily distin-guished by the presence or absence of lyso-PC. Spectraof the lysophospholipid mixtures in the presence oflyso-PC are dominated by peaks arising from this spe-cies (Fig. 7a). Moreover, no additional peaks of furtherlysophospholipids are detectable, irrespectively on thelysophospholipid composition. In the absence of lyso-PC, however, all classes of lysophospholipids are moreeasily detectable (Figs. 7b and 7c). This is in accor-dance with the previously observed higher sensitivityof MALDI-TOF mass spectrometry toward phospholip-ids of lower molecular masses (27, 29). Additionally, ifone considers the lower detection limit of MALDI-TOFmass spectrometry for intact PCs and its higher sensi-tivity toward the detection of phospholipids with lowermolecular masses, there are two characteristics ofMALDI-TOF that might explain the suppression of allother lysophospholipids in the presence of lyso-PC.However, all lysophospholipids present in the mixtureswithout lyso-PC are detectable without major prob-lems.

The spectra of PC-containing mixtures were also re-corded at a lower total lipid concentration (0.75 mg/ml)in order to check whether the dilution of a mixture canincrease the detectability of phospholipids. These re-sults were consistent with those described above, sincethe spectra were again dominated by PC and the peaksof all other phospholipids were suppressed (data notshown). Some differences were observed only in thecase of lysophospholipids. Spectra of lysophospholipidmixtures were still strongly dominated by peaks aris-ing from lyso-PC, but at lower total lipid concentra-tions, peaks corresponding to other lysophospholipidsbecame detectable. However, their intensities werevery low and were not comparable to the intensities ofthe lyso-PC peaks.

In all experiments described above the PC concen-tration in the mixtures was the same as of the otherphospholipids (0.5 mg/ml). In the next step we variedgradually the concentration of PC in the mixtures,while the concentration of other phospholipids waskept constant. The concentrations of PC in the mix-tures were 0.5, 0.25, 0.125. and 0.05 mg/ml, as indi-cated on the graph (Fig. 8). For means of comparisonthe peak height of the individual phospholipid orlysophospholipid, labeled as IPL or ILPL, respectively,was divided by the peak intensity of PC or lyso-PC(IPC or ILPC, respectively) present in the mixture.These ratios were then plotted as a function of PCconcentration. Figure 8a represents the ratio be-tween different phospholipids and PC, and Fig. 8bthe ratio between different lysophospholipids andlyso-PC. To avoid problems with overlapping peaks,we have used only peaks that differ for at least 10Da. The following peaks were analyzed: 760.6 and
496.3 (PC and lyso-PC, respectively); 476.3 (lyso-PE)

Pcen


and 740.6 (PE); 548.3 (lyso-PS) and 812.6 (PS); 455.2(lyso-PA) and 697.5 (PA); and 623.3 (lyso-PI) and885.6 (PI). All PC-containing mixtures were consid-ered and no major differences in the IPL/IPC or ILP/ILPC

ratios were found in dependence on the third phos-pholipid in the mixture. Moreover, the ratios re-mained the same when the mixture of only two phos-pholipids, i.e., PL and PC or LPL and LPC wereanalyzed. In Fig. 8 the mean values of IPL/IPC orILPL/ILPC ratios and standard deviations are pre-sented.

At the highest concentration of PC, further acidicphospholipids like PI, lyso-PS, and lyso-PA, were notdetectable at all. Lowering the PC concentrations in-creased the detectability of all other components inthose lipid mixtures. Phosphatidylinositol became de-tectable only at the lowest concentration of PC, i.e.,when the PC concentration was 10 times lower thanthat of PI (Fig. 8a). However, that might also be causedby the lower sensitivity of MALDI-TOF mass spectrom-etry toward phospholipids with a higher molecularmass (27, 29). The peak intensity of pure PI is anywaylow in comparison to other phospholipids (cf. Fig. 3),since PI has the highest molecular weight of all phos-

FIG. 7. Positive ion mass spectra of phospholipase A2 digestionA/PE/PS; and (c) PA/PS/PI. Peaks are labeled according to their

labeled with an asterisk. All phospholipids were used in a final con1.5 mg/ml).

pholipids used in this study and bears one additional

negative charge. The presence of easily detectable spe-cies, like PC, additionally decreases the sensitivity ofour detection system toward PI, and therefore, PI isobscured by the noise.

The peak intensities of all phospholipids, however,increase steadily when the concentration of PC de-creases. On the other hand, the intensities of PC orlyso-PC peaks do not decrease linearly when their con-centrations are lowered. The peak intensity remainsalmost constant and drastically decreases only at 0.05mg/ml PC concentration (data not shown). The spectraof phospholipid mixtures containing 10 times lowerconcentrations of PC (0.05 mg/ml) are characterized byrather low intensities of peaks arising from PC or lyso-PC, but only at this PC concentration remaining phos-pholipids were easily detectable.

Since the peak intensity of an individual phospho-lipid within the mixture is strongly influenced by thepeaks arising from PC or lyso-PC it is obvious that itdoes not represent the actual concentration of a givenphospholipid in the mixture. This confers serious prob-lems if a quantification of individual phospholipids isattempted by MALDI-TOF mass spectrometry withouta prior separation of the mixture into the individual

oducts of the following phospholipid mixtures: (a) PA/PC/PS; (b)/z ratios and peak identity is given in Table 3; matrix peaks aretration of 0.5 mg/ml in the mixture (total lipid concentration was

prm

phospholipid classes.

pcw ; 47( cti

214 PETKOVIC ET AL.

CONCLUSIONS

MALDI-TOF mass spectrometry has been shownpreviously to be a powerful tool for the analysis oflipids, especially for the screening of the lipid compo-sition of organic extracts of cells and tissues. The ad-vantages of MALDI-TOF mass spectrometry toward

FIG. 8. The ratios between the peak intensity of a given phosphointensities of peaks of different phospholipids (labeled as IPL) and P

eaks corresponding to different lysophospholipids (ILPL) and lyso-PConcentration of PC (in mg/ml) in the mixture. The concentration ofere analyzed: at 496.3 and 760.6, for lyso-PC and PC, respectively

lyso-PA) and 697.5 (PA); and 623.3 and 885.6, lyso-PI and PI, respe

lipid analysis are excellent signal to noise ratio, low

extent of fragmentation, and good mass resolution.Additionally, the method is fast and has only low sen-sitivity toward impurities (e.g., buffer salts) in compar-ison to other mass spectrometric methods. The mixtureof the matrix and the analyte is, in contrast to proteinsand carbohydrates, highly homogeneous since both are

d and PC as a function of PC concentration. (a) The ratio betweenPC) present in the mixtures. (b) The ratios between the intensity of

LPC). The IPL/IPC and ILPL/ILPC ratios are plotted in dependence of theer phospholipids was kept at 0.5 mg/ml. The following peak heights6.3 (lyso-PE) and 740.6 (PE); 548.3 (lyso-PS) and 812.6 (PS); 455.2vely. For peak identities cf. Table 1 and 3.

lipiC (I

(Ioth

readily soluble in organic solvents. All these advan-

1

1

1


tages of MALDI-TOF mass spectrometry make thismethod a very convenient tool for the analysis of sam-ples of biological origin.

In this paper we reported some drawbacks ofMALDI-TOF mass spectrometry for the analysis ofcrude lipid mixtures. We have shown that peaks aris-ing from different phospholipids are suppressed by PCor lyso-PC in dependence on their concentrations in themixture. Lyso-PC may even prevent the detection ofother lysophospholipids, especially acidic ones. There-fore, some caution in the interpretation of the spectrais needed if the spectra of crude lipid mixtures aredominated by peaks arising from PC or lyso-PC. Theseproblems of the detectability of different phospholipidspecies are of high importance if organic extracts ofbiological samples are analyzed, since PC is one of themost abundant species in cell membranes. In this caseit is necessary to record spectra with varying concen-trations of phospholipids.

The second problem addressed in this paper is thepotential quantification of one individual phospholipidin a lipid mixture. We have shown that quantificationof phospholipids in the presence of PC or lyso-PC is notpossible by MALDI-TOF mass spectrometry. In thepresence of PC the peak intensity does not reflect theactual abundance of phospholipids within the mixture.In spite of a number of advantages of MALDI-TOFmass spectrometry, we must state that at present aquantification of individual phospholipids in a mixtureis rather difficult and most probably prior separation ofphospholipids, e.g., by HPLC into individual classes isrequired.

On the other hand, we have shown that MALDI-TOFmass spectrometry is a suitable method for the inves-tigation of phospholipase A2 activity as well as for thedetection of lysophospholipids. These findings are ofimportance since there are not yet routine methods forthe investigation of lysophospholipids. Recently, thedetection of lysophospholipids by ESI was described(30), showing the advantages of the application of massspectrometric methods for lipid analysis. Mass spec-trometry provides an immediate information on thefatty acid composition of the corresponding lysophos-pholipid and the lipid class at the same time. Thisclearly shows the capability of MALDI-TOF mass spec-trometry for the studies of enzyme activities, as well asfor kinetic studies.

This is the first report on the different sensitivity ofMALDI-TOF mass spectrometry toward the detectionof different phospholipids in a phospholipid mixture.Our further aim is to investigate to what extent differ-ent phospholipids can be quantified. It has alreadybeen shown by our group that quantification of diacyl-glycerols by MALDI-TOF mass spectrometry can be
simply and easily performed (23).
ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsgemein-schaft (DFG-Grant AR 283/4-1, INK 23/B1-1, SFB 197, and SFB294).

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