It is usually accepted that neutral phospholipids (PLs) generate singly positively charged ions, whereas negative PLs are easily detectable in the negative ion mode when analysed by matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we demonstrate that some caution is required in the interpretation of MALDI-TOF mass spectra of PLs, since also neutral PLs have appeared to be detectable in the negative ion mode as well. Neutral and negatively charged phospholipids can generate adducts with the most commonly used matrix - 2,5-dihydroxybenzoic acid - yielding singly negatively charged ions that are detectable in the spectra. This further contributes to the complexity of the spectra and potentially leads to severe misinterpretation, particularly when unknown mixtures of PLs are analysed by MALDI-TOF MS

Arnhold, Juergen

Arnold, Klaus

Mueller, Matthias

Petković, Marijana

Schiller, Jürgen

Suess, Rosmarie

Zeitschrift für Naturforschung B

Repository of the Vinča Nuclear Institute (VinaR)

Note 331Detection of Adducts with MatrixClusters in the Positive and NegativeIon Mode MALDI-TOF Mass Spectraof PhospholipidsMarijana Petkovića,b, Jürgen Schillera,Matthias Müllera, Rosmarie Süßa, Klaus Arnolda,and Jürgen Arnholdaa Institute of Medical Physics and Biophysics, MedicalFaculty, University of Leipzig, Leipzig, Germanyb Present address: Department of Physical Chemistry,Institute of Nuclear Sciences “Vinča”, Belgrade, SerbiaReprint requests to Dr. Marijana Petković.Fax: +381 11 244 7207. E-mail: marijanapetkovic@vinca.rsZ. Naturforsch. 2009, 64b, 331 – 334;received December, 1, 2008It is usually accepted that neutral phospholipids (PLs) gen-erate singly positively charged ions, whereas negative PLsare easily detectable in the negative ion mode when analysedby matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In this study,we demonstrate that some caution is required in the inter-pretation of MALDI-TOF mass spectra of PLs, since alsoneutral PLs have appeared to be detectable in the negativeion mode as well. Neutral and negatively charged phospho-lipids can generate adducts with the most commonly usedmatrix – 2,5-dihydroxybenzoic acid – yielding singly neg-atively charged ions that are detectable in the spectra. Thisfurther contributes to the complexity of the spectra and po-tentially leads to severe misinterpretation, particularly whenunknown mixtures of PLs are analysed by MALDI-TOF MS.Key words: Phospholipids, MALDI-TOF MS, Adducts,Matrix Clusters, Positive Ion Mode, NegativeIon ModeIntroductionMatrix-assisted laser desorption and ionisationtime-of-flight mass spectrometry (MALDI-TOF MS∗)is increasingly applied for the analysis of lipids andphospholipids (PLs) [1]. Results from our labora-∗Abbreviations: MALDI-TOF MS – matrix-assisted laser de-sorption and ionisation time-of-flight mass spectrometry; PL –phospholipid; DHB – 2,5-dihydroxybenzoic acid; DPPA – 1,2-dipalmitoyl-sn-glycero-3-phosphate; DPPC – 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE – 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine; DPPS – 1,2-dipalmitoyl-sn-glycero-3-phosphoserine; PI – phosphatidylinositol; SM – sphingomyelin.0932–0776 / 09 / 0300–0331 $ 06.00 c© 2009 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.comtory [2, 3] and from the work of others [4] implythat MALDI-TOF MS can even be applied for quan-tification of lipids because of the high reproducibil-ity. Various matrices have been used for this purpose,but the most established one is 2,5-dihydroxybenzoicacid (DHB) [1]. This matrix does not interfere withpeaks arising from analytes, does not lead to markedfragmentation reactions and can be used for the ac-quisition of positive as well as of negative ion massspectra [5]. Additionally, in contrast to matrices witholefinic residues, DHB has only a weak tendency togive signals of matrix oligomers [1, 5].All lipid and PLs classes can be analysed byMALDI-TOF MS, and they are mostly detectableas singly charged ions. These ions are generated bycharge compensation and subsequent ionisation. Na+and H+ adducts are the most abundant molecularspecies since sodium ions are predominant in sam-ples of biological origin. On the other hand, H+sources are commonly added to the matrix solution,since this approach results in increased signals aris-ing from lipids [6 – 8]. In addition to their detec-tion in the positive ion MALDI-TOF mass spectra,acidic PLs can be detected as singly negatively chargedions.We have previously reported that PLs with a cholinehead group – phosphatidylcholines (PC) and sphin-gomyelins (SM) – can generate clusters with one nega-tively charged DHB molecule [9]. Negatively chargedclusters generated in that way can then be easily de-tected and analysed by mass spectrometry. This obser-vation was made with lipoproteins that contain hugeamounts of PC and SM.In the present report, we demonstrate that additionalcaution is required in the spectra interpretation, be-cause also other PL species – neutral as well as neg-atively charged ones – can generate clusters with DHBas such or with sodiated DHB (DHB : Na) providingmore complex peak patterns than previously assumed.The intensity of these PL : matrix clusters seems to de-pend on the purity of the applied DHB, and particularlyon the content of inorganic salts in the solvents used formatrix and/or lipids.Results and DiscussionIn this report, we demonstrate that neutral and acidicPLs can generate adducts with the DHB matrix, which332 NoteFig. 1. Positive ion MALDI-TOF mass spectra of DPPA (a),DPPC (b), DPPE (c), DPPI (d) and DPPS (e). All spec-tra of the PLs were recorded with DHB as matrix. Peaksare labelled according to their m/z ratios, and their identityis indicated. “M” indicates the completely dissociated PLmolecule, and characteristic matrix peaks are indicated byan asterisk.contributes to the complexity of the spectra and canlead to severe misinterpretation.Fig. 1 shows the positive ion MALDI-TOF massspectra of DPPA (a), DPPC (b), DPPE (c), DPPI (d),and DPPS (e). Peaks are labelled according to theirm/z ratio, and their identity is indicated in the Figure.In general, neutral PLs require only one cation (forcationisation) to be detectable as positive ions [1, 6 –8]. On the other hand, acidic PLs first require neu-tralisation with the adequate number of cations (de-pending on their charge) and the addition of onemore cation in order to be detected in the posi-tive ion mode MALDI-TOF mass spectra. These PLsare more easily detected as negative ions. Peaks thatarise from characteristic matrix adducts (labelled withan asterisk in Fig. 1) are described in detail else-where [7]. “M” corresponds to the molecular massof the completely dissociated PL molecule, whereas“DHB : Na” indicates clusters of DHB with one H+exchanged by a sodium ion. This molecule thusremains uncharged. The addition of one DHB:Naproduces a PL-matrix adduct with mass differenceof 176 Da compared to a completely sodiated PL (cf.below).The spectra of each of the above mentioned PLswere discussed in detail earlier [1, 6 – 8], and here thespectra characteristics will only be shortly summarisedwith the emphasis being on adducts with the matrix, ormatrix cluster(s).DPPA (1a) bears two negative charges, and aftercharge compensation with two cations and ionisationwith a further one this PL is detectable at m/z = 693.5,715.5 and 737.5 (corresponding to the addition of 2H+ and Na+, H+ and 2 Na+ as well as 3 Na+, respec-tively). In the positive ion MALDI-TOF mass spectraof DPPA, cluster adducts were not observed. Peaks athigher m/z are characteristic matrix peaks [7]. The pos-itive ion MALDI-TOF mass spectrum of DPPC (1b)contains two peaks (at m/z = 734.6 and 756.6) thatare generated by the cationisation of DPPC either witha proton or a sodium ion, respectively. The additionof a cluster DHB : Na to DPPC results in the peak atm/z = 932.6 DPPE (1c) yields more complex posi-tive MALDI-TOF mass spectra in comparison to PCbecause the ethanolamine head group possesses anexchangeable proton. Thus, DPPE yields three peaks(m/z = 692.5, 714.5 and 736.5) corresponding to thecharge compensation with H+, Na+ and the replace-ment of H+ by Na+, respectively. Also in this case,the DPPE-matrix cluster (the peak at m/z = 912.5) isgenerated only from the completely sodiated form ofthis PL.In (1d) and (1e) the positive ion MALDI-TOF massspectra of DPPI and DPPS, respectively, are given.These singly charged acidic PLs require charge com-pensation with one cation and ionisation with a fur-ther one to be detectable in positive ion mode. DPPIis detected at m/z = 833.5 and at 855.5, correspondingto the addition of H+, Na+ and 2 Na+, respectively,whereas the spectrum of DPPS contains the followingpeaks at m/z = 736.5, 758.5 and 780.5, correspondingto the charge compensation with 2 H+, H+ and Na+,and 2 Na+, respectively. Moreover, with DPPS an ex-change of the proton by a sodium ion on the head groupyields one further peak at m/z = 802.5. PI as well as PSgenerate also clusters with DHB : Na, yielding peaks atm/z = 1031.5 in the spectrum of DPPI (1d) and at m/z =1154.5 in the positive ion mass spectrum of DPPS (1e).Only for the completely sodiated forms of both of theseNote 333Fig. 2. The ratio between peak intensity of thecompletely sodiated form of the PL and of thepeak intensity of the matrix clusters is given in (a);in (b) the signal-to-noise ratio of the peak arisingfrom the cluster ion [POPC+Na++DHB:Na+]+and of the ion [SOPS–H++3Na++DHB:Na+]+ inrelationship with the concentration of the lipid isshown. All spectra were recorded with DHB ma-trix, under delayed extraction condition and in thereflector mode. Presented results are the averageof four spectra recorded.Table 1. Identity of peaks detected in the negative ionMALDI-TOF mass spectra of PLs.Peak position, m/z ratio Peak identity647.5 [DPPA2−+H+]−734.5 DPPS−809.5 DPPI−823.5 [DPPA2−+H++DHB−+Na+]−866.5 [DPPE+–H++DHB−+Na+]−910.5 [DPPS−+DHB−+Na+]−932.5 [DPPS−–H++Na++DHB−+Na+]−985.5 [DPPI−+ DHB−+Na+]−999.5 [DPPA2−+H++2(DHB−+Na+)]−1042.6 [DPPE+–H++2(DHB−+Na+)]−PLs, the addition of DHB : Na is leading to detectablepeaks.MALDI-TOF mass spectra of PLs were alsorecorded in the negative ion mode, and the detectedpeaks with their identities are given in Table 1. It isimportant to note the following: i) acidic PLs, suchas DPPA, DPPS and DPPI, which were used in thisstudy, require a lower number of cations in compari-son to the positive mode to be detectable as negativeions; ii) neutral PLs are easily detectable as positiveions [6, 7], whereas in the negative ion mode they aredetectable exclusively as clusters either with negativelycharged DHB or with DHB : Na, in dependence on thenature of the head group. This behaviour, however, hasbeen demonstrated in our previous work for choline-containing PLs [8].Relative tendencies of various lipid species to pro-duce matrix adducts that were detectable by MALDI-TOF MS in the positive ion mode are presented inFig. 2(a). The ratio of the peak intensities of the com-pletely sodiated form of DPPC, DPPS, DPPE andDPPA and the matrix cluster is presented. The inten-sity of the completely sodiated form of acidic PLs islower compared to the intensity of the peak generatedby the addition of a matrix cluster, at least as far as it isdetectable in the positive ion mode. The opposite resultis obtained for neutral PLs. Therefore, the acidic PLstested in this study, DPPS and DPPA, show somewhathigher tendency to generate adducts with matrix clus-ters in comparison to neutral PLs (DPPC and DPPE).To check whether the concentration of an analyteaffects the intensity of peaks arising from adductsof PLs with the matrix, positive ion MALDI-TOFmass spectra were recorded at various concentra-tions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPC). Results of these experimentsare shown in Fig. 2(b), and it can be observed thatthe signal intensities increase with increasing PL con-centration. A signal arising from the matrix adductof SOPC was not detectable at the lowest concentra-tion of this PL, most probably due to already observedhigher detection limits for acidic PLs, i. e. higher con-centrations of them are required for their detection inthe spectra in comparison to the neutral ones [10]. Onthe other hand, the intensity of the matrix adduct ofPOPC was rather high even at the lowest concentra-tion. At highest concentrations tested, the intensity ofsignals arising from acidic PLs was comparable, evensomewhat higher than the intensity of signals arisingfrom POPC. Finally, by this experiment we have testedwhether the generation of adducts with matrix clustersis related exclusively to PLs with saturated fatty acidresidues. It has been shown that adducts with matrixcluster(s) are observable also in the case of PLs withunsaturated fatty acid residue(s).In summary, we have demonstrated that differentPLs can generate complex adducts with the DHB ma-trix and/or matrix clusters, thus leading to complexpositive and negative ion MALDI-TOF mass spectraof PLs. The addition of one or, in some cases, twoDHB : Na clusters was observed in the positive ionMALDI-TOF mass spectra of PLs – except in the caseof highly acidic PA – as well as in the negative ionmode mass spectra of acidic PLs. Peaks generated by334 Notethe addition of one negatively charged DHB moleculewere observed in the negative ion MALDI-TOF massspectra of neutral PLs – DPPC and DPPE. The ap-pearance and intensity of the peaks corresponding tothese cluster ions cannot be completely explained atthis stage. However, it might be that these additionalpeaks decrease the intensity of the PL peaks of inter-est, thus enhancing the risk of their misinterpretation.This risk is particularly high when unknown lipid/PLmixtures of biological origin are analysed by MALDI-TOF MS. To our knowledge, generation of PL-DHBclusters is favoured by (i) a high ion concentration and(ii) enhanced laser intensities. The tendency of variouslipids to generate adducts with the matrix will be fur-ther investigated, and this report is a preliminary warn-ing to those who use DHB/trifluoroacetic acid for thelipid analysis by MALDI-TOF MS.Experimental SectionChemicalsMatrix for MALDI-TOF mass spectrometry – 2,5-dihydroxybenzoic acid, DHB – as well as solvents –chloroform and methanol (with less than 0.00005 %of sodium and potassium) were purchased from Fluka(Neu Ulm, Germany). Phospholipids: 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) were obtained from AvantiPolar Lipids Inc. (Alabaster, Alabama, USA) Phosphatidyli-nositol (1,2-dipalmitoyl-sn-glycero-phosphoinositol, DPPI)was purchased from Echelon Research Laboratories (SaltLake City, Utah, USA). These chemicals were of highestcommercially available purity and were used without furtherpurification.Preparation of samples for MALDI-TOF MSPLs used in this study were applied as chloroform solu-tions at various concentrations. A small volume (1.2 µL) ofa PL solution was applied onto the sample plate and imme-diately dried under a moderately warm stream of air. After-wards, the same volume of a matrix solution (0.5 M DHBin methanol) was added over a lipid film and also immedi-ately dried. This approach was shown to improve the homo-geneity of the sample/analyte mixture and the reproducibilityof the spectra. In some experiments, DHB was dissolved inmethanol saturated with NaCl or KCl.MALDI-TOF MSMALDI-TOF mass spectra were acquired on a VoyagerBiospectrometry workstation (Perseptive Biosystems, Flam-ingham, MA, USA). The system utilises a pulsed nitrogenlaser emitting at 337 nm. The pressure in the ion chamberwas maintained between 1×10−7 and 1×10−4 Torr. Otherconditions were: delayed extraction (DE), accelerated volt-age of 20 kV and reflector mode, so that the total field-freetime-of-flight distance was 2 m. These conditions signifi-cantly improved mass resolution and mass accuracy. An in-ternal calibration was performed by setting the peak of theprotonated DHB to its appropriate value (155.034 Da). Laserintensity was kept about 10 % over the threshold to obtainthe best signal-to-noise ratio. All lipid spectra were acquiredusing a low-mass gate at 400 Da to prevent the detector fromsaturation by ions arising from matrix degradation or photo-chemical reactions.AcknowledgementsThis work was supported by the German Research Coun-cil (DFG Schi 476/5-1) and the Federal Ministry of Educa-tion and Research (Grant BMBF 0313836).[1] J. Schiller, R. Süß, J. Arnhold, B. Fuchs, J. Leßig,M. Müller, M. Petković, H. Spalteholz, O. Zschörnig,K. Arnold, Prog. Lipid Res. 2004, 43, 449 – 488.[2] M. Petković, J. Schiller, J. Müller, M. Müller, K. Ar-nold, J. Arnhold, Analyst. 2001, 126, 1042 – 1050.[3] S. Benard, J. Arnhold, M. Lehnert, J. Schiller, K. Ar-nold, Chem. Phys. Lipids 1999, 100, 115 – 120.[4] G. R. Asbury, K. Al-Saad, W. F. Siems, R. M. Hannan,H. H. Jill, J. Am. Soc. Mass Spectrom. 1999, 10, 983 –991.[5] J. Schiller, R. R. Süß, B. Fuchs, M. Müller, M. Pet-ković, O. Zschörnig, H. Waschipky, Eur. Biophys. J.2007, 36, 517 – 527.[6] J. Schiller, J. Arnhold, S. Benard, M. Müller, S. Reichl,K. Arnold, Anal. Biochem. 1999, 267, 45 – 56.[7] M. Petković, J. Schiller, M. Müller, S. Benard, S. Re-ichl, K. Arnold, J. Arnhold, Anal. Biochem. 2001, 289,202 – 216.[8] D. J. Harvey, Mass Spectrom. 1995, 30, 1333 – 1346.[9] J. Schiller, R. Süß, M. Petković, O. Zschörnig,K. Arnold, Anal. Biochem. 2002, 309, 311 – 314.[10] M. Müller, J. Schiller, M. Petković, W. Oehrl, R. Einze,R. Wetzker, K. Arnold, J. Arnhold, Chem. Phys. Lipids2001, 110, 151 – 164.

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Detection of Adducts with Matrix Clusters in the Positive and Negative Ion Mode MALDI-TOF Mass Spectra of Phospholipids

Detection of Adducts with Matrix Clusters in the Positive and Negative Ion Mode MALDI-TOF Mass Spectra of Phospholipids

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