Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Chemistry and Physics of Lipids

110 (2001) 151–164

Limits for the detection of (poly-)phosphoinositides bymatrix-assisted laser desorption and ionization time-of-flight

mass spectrometry (MALDI-TOF MS)

Matthias Muller a,*, Jurgen Schiller a, Marijana Petkovic a, Wolf Oehrl b,Regina Heinze b, Reinhard Wetzker b, Klaus Arnold a, Jurgen Arnhold a

a Institute of Medical Physics and Biophysics, Medical Faculty, Uni�ersity of Leipzig, Liebigstr. 27, D-04103 Leipzig, Germanyb Research Unit ‘Molecular Cell Biology’, Medical Faculty, Friedrich-Schiller-Uni�ersity Jena, Drackendorfer Str. 1,

D-07747 Jena, Germany

Received 30 October 2000; received in revised form 27 December 2000; accepted 15 January 2001

Abstract

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has beenrecently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for thedetection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and ahigh phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as theminimum of analyte on the sample plate resulting in a signal-to-noise-ratio of S/N=5. Using 2,5-dihydroxybenzoicacid (DHB) as matrix the detection limit for phosphatidylinositol (PI) is seven times higher than for phosphatidyl-choline (PC) and further increases with increasing phosphorylation or in mixtures with other well-detectablephospholipids. For phosphatidylinositol-tris-phosphate (PIP3) in a mixture with PC, the limit is about 20 times higherthan for PI. The consequences for the experimental conditions are discussed. It is advisable to pre-separate PPI frombiological lipid mixtures prior to the application of MALDI-TOF MS. © 2001 Elsevier Science Ireland Ltd. All rightsreserved.

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Abbre�iations: DHB, 2,5-dihydroxybenzoic acid; DPPC, 1,2-dipalmitoyl-sn-3-glycero-phosphocholine (dipalmitoylphosphatidyl-choline); DPPI, 1,2-dipalmitoyl-sn-3-glycero-phosphoinositol (dipalmitoylphosphatidylinositol); DPPIP3, 1,2-dipalmitoyl-sn-3-glyc-ero-phosphoinositol-3,4,5-phosphate (dipalmitoylphosphatidylinositoltrisphosphate); EDTA, ethylenediaminetetraacetic acid; ESI,electrospray ionization; FAB, fast atom bombardment; MALDI-TOF, matrix-assisted laser desorption and ionization time-of-flight;MS, mass spectrometry; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI3K, phosphoinositide3-kinase; PIP, phosphatidylinositol-4-monophosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PPI, (poly-)phosphoinositides; PS, phosphatidylserine; SAPIP3, 1-stearoyl-2-arachidonoyl-sn-3-glycero-phosphoinosi-tol-3,4,5-trisphosphate (stearoyl-arachidonoyl-phosphatidylinositoltrisphosphate); TFA, trifluoroacetic acid; THA,2,4,6-trihydroxyacetophenone.

* Corresponding author. Tel.: +49-341-9715720; fax: +49-341-9715709.E-mail address: [email protected] (M. Muller).

0009-3084/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0009 -3084 (01 )00132 -3

M. Muller et al. / Chemistry and Physics of Lipids 110 (2001) 151–164152

Keywords: Phosphoinositides; Lipid analysis; Detection limits; MALDI-TOF mass spectrometry

1. Introduction

(Poly-)phosphoinositides (PPI) represent an im-portant group of phospholipids, although phos-phatidylinositol (PI) and its phosphoryl-ated derivatives phosphatidylinositol-4-phosphate(PIP) and phosphatidylinositol-4,5-bisphosphate(PIP2) constitute only about 5% of total cellularlipids. PPI do not primarily play a structural rolein cellular membranes, but are crucial for cellularsignaling. Thus, the hydrolysis of phosphoinosi-tides by phospholipase C enables the transductionof extracellular signals through the plasma mem-brane by the formation of second messengers(Berridge and Irvine, 1984; Divecha and Irvine,1995). PPI are involved in the control of diversecellular functions including proliferation as wellas endo- and exocytosis (Isenberg, 1991; Janmey,1995; Duckworth and Cantley, 1996; Isenberg andNiggli, 1998). Phosphoinositides that are phos-phorylated at the third position of the inositolring, like phosphatidylinositol-3,4,5-trisphosphate(PIP3), are of special importance. These lipids aregenerated by phosphoinositide 3-kinases (PI3K)and are assumed to possess multiple functions.For example, they are involved in the oxidativeactivity of neutrophils, the intracellular proteintransport and the cell growth (Kapeller and Cant-ley, 1994; Stoyanov et al., 1995; Carpenter andCantley, 1996; Zvelebil et al., 1996; Wymann etal., 1996).

Physiological, biochemical and biophysicalstudies on the role of phosphoinositides in cellularmembranes need reliable analytical data on thepresence as well as the concentration of theselipids. The application of mass spectrometricmethods is useful, especially due to their highsensitivity. However, the effective applicability ofmass spectrometry depends on the proper choiceof the corresponding MS technique. Althoughnowadays a number of different ionization tech-niques is available, only three of them play amajor role in the field of phospholipid analysis:fast atom bombardment (FAB) (Sherman et al.,1985; Jensen et al., 1987), electrospray ionization

(ESI) (Kerwin et al., 1994; Spickett et al., 1998;Jerlich et al., 2000), and matrix-assisted laser des-orption ionization (MALDI). FAB was success-fully applied for the detection ofphosphoinositides by Sherman et al. (1985). ESIwas applied for the analysis of oxidation productsof phospholipids (Spickett et al., 1998) and alsofor the structural characterization as well as thequantification of phosphoinositides (Michelsen etal., 1995; Gunnarsson et al., 1997; Hsu and Turk,2000). The use of ‘‘hard’’ ionization methods likeFAB requires high experience in the analysis ofmolecule fragmentation patterns, since the molec-ular ion is often not detectable. Therefore, FAB islimited towards its use in mixture analysis. Incontrast, MALDI as well as ESI have the advan-tage that no derivatization of the sample is re-quired and that the ionization is ‘soft’. ESI can bevery easily coupled with liquid chromatography toseparate sample components prior to their massspectrometric analysis. This cannot be done soeasily with MALDI. However, MALDI-TOF MSon lipids has the advantage that all experimentscan be performed in a single organic phase, sinceboth, the lipid as well as the matrix (e.g. DHB)are readily soluble in organic solvents. This pro-vides extremely homogeneous matrix/analytecrystals and leads to an excellent reproducibility–especially in comparison to the mass spectromet-ric analysis of proteins. Additionally, MALDI ismore sensitive than ESI and is not affected byimpurities to such a high extent. In a recent paper,we demonstrated for the first time that theseproperties allow the efficient and convenient de-tection of lipids by MALDI-TOF MS (Schiller etal., 1999a). The technique was successfully usedfor the analysis of lipids in model membranes(Harvey, 1995; Marto et al., 1995) as well as inorganic cell extracts (Schiller and Arnold, 2000).Additionally, MALDI-TOF MS allows the detec-tion of components in a lipid mixture qualitatively(Schiller et al., 2000). Using a known internalstandard, the quantification of lipids (e.g. diacyl-glycerols) is also possible (Benard et al., 1999),even if the molecular weight influences signal in-tensity (Asbury et al., 1999).

M. Muller et al. / Chemistry and Physics of Lipids 110 (2001) 151–164 153

The chemical structure of a certain molecule,however, strongly affects its detectability byMALDI-TOF MS. Phosphoinositides differ fromother phospholipids by their headgroup structurecontaining the inositol ring system as well as bytheir charge state. While PI bears one negativecharge at the dissociated phosphodiester group,additional phosphomonoester groups at the inosi-tol ring lead to higher negative charges for PIP(z= −3) and PIP2 (z= −5) at pH 7.4 (Toner etal., 1988; Gabev et al., 1989). The charge of PIP3

is still higher (z= −7 at pH 7.4). Furthermore,phosphoinositides possess a higher molecularmass than all other known phospholipids contain-ing two acyl chains (more complex lipid structureslike cardiolipin are not considered here). The bio-logically relevant mass region of phosphoinosi-tides ranges from about 880 Da to about 1120Da.

In this study, the detectability of phosphoinosi-tides by MALDI-TOF mass spectrometry wasinvestigated. We used the signal-to-noise-ratio todescribe the quality of a given spectrum and thedetectability of a certain phosphoinositide. Wedefined the minimum amount of analyte to obtaina reasonable signal-to-noise-ratio as detectionthreshold. It will be shown that the molecularproperties of the PPI are responsible for PPI-de-tection limits, which are by far higher than formore abundant cellular lipids, e.g. phosphatidyl-cholines. Since it is known that the quality ofMALDI-TOF MS data strongly depends on thechosen matrix system and on the way of samplepreparation, different approaches to improve thesample preparation technique are also comparedwith respect to the detectability of phosphoinosi-tides. Finally, consequences for the application ofMALDI-TOF MS to investigations on phospho-inositides in model and biologically relevant sys-tems are discussed.

2. Materials and methods

2.1. Chemicals

Phosphatidylcholine (PC) from egg yolk, 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and the

ammonium salt of bovine liver phosphatidylinosi-tol (PI) were purchased from Avanti Polar Lipids(Alabaster, Alabama, USA). The sodium salt of1,2-dipalmitoyl-phosphatidylinositol (DPPI) wasfrom CPS Chemie+Service GmbH (Duren, Ger-many). The sodium salts of phosphatidylinositol-4-phosphate (bovine brain, PIP) andphosphatidylinositol-4,5-bisphosphate frombovine brain (PIP2) were obtained from Sigma(Deisenhofen, Germany). The supplier declaredthat both lipids consist mainly of stearic andarachidonic acid chains. The ammonium salt of1-stearoyl-2-arachidonoyl-phosphatidylinositol-3,4,5-trisphosphate (SAPIP3) was purchased fromAlexis Deutschland (Grunberg, Germany). Theammonium salt of 1,2-dipalmitoyl-phosphatidyli-nositol-3,4,5-trisphosphate (DPPIP3) was ob-tained from Biomol (Hamburg, Germany).

Lipids delivered as powders were dissolved inchloroform. For the PPI, the addition ofmethanol and a few drops of HCl were required.All solvents (chloroform, methanol) were ofHPLC grade and obtained from Fluka Fein-chemikalien GmbH (Neu-Ulm, Germany). Trifl-uoroacetic acid (TFA) and the matrix substances2,5-dihydroxybenzoic acid (DHB) and 2,4,6-trihy-droxy-acetophenone (THA) were obtained fromthe same manufacturer. Spermine tetrahydrochlo-ride was from Sigma (Deisenhofen, Germany).

2.2. Sample preparation

The following standard technique for thepreparation of MALDI-TOF MS samples wasused if not otherwise stated: Lipid stock solutionsof the appropriate concentration were directlyapplied to the sample plate as 1 �l droplet. Afterthe solvent was evaporated within 5–10 s under agentle stream of warm air, 1 �l of the matrixsolution (0.5 M 2,5-dihydroxybenzoic acid (DHB)solution in methanol containing 0.1% trifl-uoroacetic acid (TFA)) was added to the plateand samples were allowed to co-crystallize. Dry-ing the samples with a moderate warm stream ofair extremely improved the homogeneity ofcrystallization.

Alternatively, the appropriate amounts of lipidstock solutions and the DHB solution were pre-


mixed in a small glass vial and 2 �l of the result-ing mixture were applied for the analysis.

To check the influence of the sample prepara-tion conditions, in some cases the TFA-content ofthe matrix solution was varied or the lipid stocksolutions were prepared in organic solutions con-taining CaCl2, NaCl, spermine tetrahydrochlorideor EDTA. Clear solutions (1 mM) of these salts ina mixture of chloroform, methanol and 1 M HCl(2:1:0.02, v/v/v) were obtained by intense vortex-ing and overnight storage at room temperature.

2.3. MALDI-TOF mass spectrometry

All MALDI-TOF mass spectra were acquiredon a Voyager Biospectrometry DE workstation(PerSeptive Biosystems, Framingham, MA). Thesystem utilizes a pulsed nitrogen laser, emitting at337 nm. The pressure in the ion chamber was heldbetween 1×10−7 and 4×10−7 Torr. All mea-surements were done under delayed extractionconditions (DE-mode), improving both mass ac-curacy and mass resolution (Bahr et al., 1997).The extraction voltage was 20 kV. A ‘low-mass-gate’ was set at m/z=500 to prevent the satura-tion of the detector by ions resulting from thematrix (Hillenkamp et al., 1991). To enhance thespectral resolution all spectra were recorded in thereflector mode. The instrument provides a field-free time-of-flight distance of 2 m in that mode.An internal mass calibration was done in thepositive ion mode by setting the base peak of thematrix substance DHB to its appropriate value of155.034 Da. For each mass spectrum 128 singlelaser shots were averaged. Although not alwaysshown, both positive and negative ion mass spec-tra were acquired in all cases.

There are primarily two features of samplepreparation and measuring setup that have a ma-jor impact on the quality of MALDI-TOF massspectra: The homogeneity of the matrix/samplemixture and the applied laser power. The firstparameter was controlled empirically. Most or-ganic stock solutions of lipids and matrix sub-stances are easily miscible under the describedconditions and yield a very homogeneous co-crys-tallization of the sample with the matrix (if notindicated otherwise). The applied laser power was

controlled carefully, because it has a great influ-ence on the signal intensity (Benard et al., 1999).In general, the laser power was kept about 10%over threshold to obtain the best signal qualitywith low noise. To avoid influences of laser powerinstabilities, a calibration measurement and finetuning of the laser power using a 1,2-dipalmitoyl-phosphatidylinositol sample as standard was donefrom time to time to maintain reproducibility.

2.4. Calculation of the signal-to-noise ratio anddetection limits

The signal-to-noise-ratio (S/N) was calculatedfrom Eq. (1) as demonstrated in Fig. 1.

SN

=2.5SH−0.5NPP

NPP

(1)

Here, SH is the signal height at the center of acertain mass peak over the lower limit of noiseand NPP is the peak-to-peak noise amplitude,determined from the maximum and minimum val-ues of 250 points of pure noise (Gunther, 1983;Braun et al., 1998). For optimized standard sam-ples, which result in peak heights near the satura-tion level of the detector, maximum S/N ratios ofabout 500–600 were obtained. The value of S/N=5 was taken as the lowest threshold limit to

Fig. 1. Calculation of the S/N-ratio: The values SH (signalheight of a certain peak over the lower limit of noise) and NPP

(the peak-to-peak noise amplitude) used in Eq. (1) are marked.The figure shows the spectrum of dipalmitoylphosphatidyl-choline (DPPC). The spectrum was recorded in the positiveion mode using 136 pmol of lipid on the sample plate. Thepeaks represent the molecular ion (M+H)+ at m/z=734.6Da and the first sodium adduct (M+Na)+ at m/z=756.6 Da.The calculated S/N-ratio at m/z=734.6 is about 200.


clearly distinguish any peak as a signal from thenoise. The lipid concentration needed to obtainS/N=5 was defined as detection limit.

3. Results

3.1. Mass spectra of phosphoinositides

Fig. 2 shows the negative ion MALDI-TOFmass spectra of selected phosphoinositides withdifferent degrees of phosphorylation at the inosi-tol ring: PI (a), PIP (b), PIP2 (c), dipalmitoyl-PIP3

(DPPIP3, d) and stearoyl-arachidonoyl-PIP3

(SAPIP3, e). While the latter two substances weresynthetic phospholipids, PI, PIP, and PIP2 were ofnatural origin. However, their spectra correspondto a single fatty acid composition: one stearoyland one arachidonoyl residue. Other fatty acidcompositions were not detectable.

PI (18:0, 20:4) yields the isotopically resolvedmolecular ion peak at m/z=885.5 Da. This peakcorresponds to the exact molecular mass (calcu-lated with atomic masses of the most abundantisotopes: 1H, 12C, 16O, 31P) of the stearoyl-arachi-donoyl-PI without further addition of anycations. We suppose that the single negativecharge of the PI ion leads directly to the peak,whereby no further addition of ions is required(Schiller et al., 1999a).

With an increasing number of phosphategroups the peaks of the corresponding molecularion are shifted to higher m/z ratios. These differ-ences can be explained by two contributions: (i)the substitution of a hydroxy group by a phos-phate group, and (ii) the required compensationof the corresponding negative charges (Schiller etal., 1999a). Briefly, assuming that all peaks shownin Fig. 2 represent species carrying one negativecharge, the peaks can be interpreted consideringthe need to compensate the higher negative chargeof the lipid by the addition of protons or sodiumions. The most expressed peaks in all spectra arecaused by charge compensation by protons only.Two protons are involved in the case of PIP(m/z=965.5, b), four in PIP2 (m/z=1045.5, c),and six in PIP3 (m/z=1049.9 for DPPIP3, d, andm/z=1125.4 for SAPIP3, e).

Mass spectra of higher phosphorylated lipidsexhibit additional peaks at m/z ratios that differby 22 or 44 Da from that of the protonatedmolecular ion. Here, one or two protons arereplaced by sodium. Since TFA was present in thematrix solution, charge compensation is mainlydue to the addition of protons and only to asmaller extent by sodium. However, altering theratio of cations in solution would also alter thepeak ratio between proton and sodium adducts.In the SAPIP3 spectrum (Fig. 2e), the peak atm/z=1097.4 most likely corresponds to a con-tamination of the sample with PIP3 containing apalmitoyl instead of a stearoyl residue. In allcases, fragmentation reactions occur only to anegligible extent and the yield of multiply chargedions was below the detection limit (data notshown).

The asterisks in Fig. 2 label the peaks that wereused to calculate the signal-to-noise ratios for PI,PIP, PIP2 and SAPIP3 given in the followingsections.

3.2. Detectability of phosphoinositides depends onthe phosphorylation state

The signal-to-noise ratio in MALDI-TOFmass spectra of phosphoinositides was investi-gated as a function of the amount of lipid on thesample plate. Fig. 3 shows the S/N valuescalculated from the positive ion mass spectra ofPI, PIP, PIP2, and phosphatidylcholine (dipalmi-toyl, DPPC). The corresponding positiveion mass spectrum of DPPC is shown in Fig. 1.DPPC has been chosen for means of comparison,because it gave only the protonated molecular ionpeak at m/z=734.6 Da and the sodiumadduct peak at m/z=756.6 Da. The peak atm/z=734.6 Da was used for the S/N calculationfor DPPC.

The S/N values for all measured lipids decreasewith lowering lipid amount. In general, the S/Nvalues for phosphoinositides in the positive ionmode were much lower than that for PC at com-parable amounts. While the minimum thresholdamount (S/N=5) for PC in the positive ion modeis about 3�1 pmol, the detection limits for phos-phoinositides are 67.5�11 pmol for PI, 143�38


Fig. 2. Negative ion MALDI-TOF mass spectra of phosphoinositides: PI from bovine liver (a), PIP (b), and PIP2 (c) from bovinebrain, dipalmitoyl-PIP3 (d) and stearoyl-arachidonoyl-PIP3 (e). Samples were prepared in a matrix of dihydroxybenzoic acid (0.5 Min methanol). The lipid amounts on the plate were 113 pmol for PI, 261.8 pmol for PIP, 478 pmol for PIP2, 572 pmol for DPPIP3,and 444.3 pmol for SAPIP3. Spectra are the average of 128 laser shots. Asterisks indicate the peaks used for the calculation of thesignal-to-noise ratio.

pmol for PIP, and 328.4�92 pmol for PIP2. PIP3

was not detectable with a sufficient signal to noiseratio in the positive mode under our experimentalconditions.


In Fig. 4, the concentration dependence of theS/N values is shown for the negative ion massspectra of PI, PIP, PIP2, and PIP3. The S/N ratiodecreases with decreasing phospholipid amountfor all investigated lipids. S/N depends fairly lin-early on the lipid quantity over a wide range in adouble logarithmic scaling. Below a certainthreshold that is different for each lipid, the signaldisappears nearly completely (S/N about 2.5). Itis obvious that these thresholds for phosphoinosi-tides are much lower in the negative than in thepositive ion mode (cf. Fig. 3).

Comparing the different phosphoinositides, aclear dependence of their detectability on thephosphorylation state is found. The higher thenumber of phosphate groups, the lower is thequality of the corresponding spectrum. Thethreshold lipid amount defined as detection limitincreases with increasing phosphorylation. Thedetection limits for PI, PIP, PIP2, and PIP3 aregiven in Table 1. Additionally, the detection limit

Fig. 4. Signal-to-noise ratio as a function of the phospholipidamount on the plate for MALDI-TOF negative ion massspectra of PI (�), PIP (�), PIP2 (�), and SAPIP3 (�). Allconditions were as in Fig. 3. For better clarity not all errorbars are drawn.

Fig. 3. Signal-to-noise ratios as a function of the phospholipidamount on the plate for MALDI-TOF positive ion massspectra of phosphatidylcholine (DPPC, �), PI (�), PIP (�),and PIP2 (�). 2,5-Dihydroxybenzoic acid (0.5 M in methanol)containing 0.1 vol.% TFA was used as matrix. Data pointsrepresent the mean of six measurements (two different prepa-rations with three measurements each), error bars representthe S.D. The line at S/N=5 represents the threshold definedas the detection limit.

for DPPC in the positive ion mode is shown forcomparison. This lipid is not detectable as nega-tive ion (Schiller and Arnold, 2000).

3.3. Lipid signals decrease in lipid mixtures

To model the situation in biological systems,the detectability of PIP2 and PIP3 was examinedin mixtures with PC as a representative examplefor the most abundant phospholipids in cellularmembranes.

Fig. 5 shows the PIP2 concentration depen-dence of S/N values obtained from the negativeion mass spectra of samples prepared from differ-ent PIP2/PC mixtures. Qualitatively, the depen-dence of S/N on the PIP2 concentration is alsomaintained irrespectively on the PC concentrationin the mixtures. However, the curves are clearlyshifted to higher PIP2 concentrations in the pres-ence of PC in comparison to pure PIP2. Thisindicates that the detectability of PIP2 is reducedby the presence of PC. While PIP2 alone is welldetectable above 123.4�14.6 pmol, the detectionlimit is 156�47 pmol in the presence of 100 ng


(132 pmol) PC. The detection limit for PIP2 in-creases to 273.5�68 pmol or 424.3�91 pmol inthe presence of 500 ng (658 pmol) or 1000 ng(1315 pmol) PC, respectively. This holds althoughPC is not detectable as a negative ion. In all cases,the positive ion mass spectra exhibit well-resolvedPC signals with intensities near the saturationlimit of the detector independently of the PIP2

concentration (spectra not shown).The corresponding results for PIP3/PC mixtures

are given in Fig. 6. In addition to the fact that thedetection limit for PIP3 alone is by far higher thanfor all other investigated phospholipids, the pres-ence of PC further decreases the PIP3 detectabil-ity. The measured threshold values for PIP3 are334�22 pmol in the presence of 100 ng (132pmol) PC, and 400�16 pmol in the presence of500 ng (658 pmol) PC. Furthermore, it was notpossible to detect PIP3 in a ternary mixture ofdifferent amounts of PIP3 with PIP2 (95.6 pmol)and PC (658 pmol) up to PIP3 amounts of 500pmol. From the extrapolation of the appropriateS/N dependence to higher concentrations in Fig.6, one can roughly estimate a detection limit of atleast 700 pmol for PIP3 in this case.

3.4. Influence of sample preparation

In the preceding sections it was demonstratedthat the PPI phosphorylation state determines thedetectability of these lipids by MALDI-TOF MS.To check, whether the detectability of PPI can beinfluenced by the presence or absence of positiveor negative ions in the lipid/matrix mixture, thesample preparation was performed in that waythat either positive ions were added (Ca2+, Na+,spermine4+) or EDTA was added in order toremove cations. The results of representative mea-surements carried out with PIP2 as a test sub-stance are given in Table 2.

In general, there is no significant improvementof the PPI detectability when cations are added tothe sample. Although the detection limit is de-creased in some cases (e.g. the four times posi-tively charged spermine reduces the detection limitfor PIP2 to 89.6�17.2 pmol), it is still compara-bly high. The addition of Ca2+ or Na+ evenincreases the detection limit for PIP2. In the caseof Na+, the relative height of the sodium adductpeaks increases drastically. Therefore, the firstsodium adduct of PIP2 (m/z=1067.1 Da) was

Table 1Detection thresholds for different lipids

Phospholipid Detection limit

Amount of lipid on the sample plate Concentration of stock solution Lipid per matrix (10−5

(pmol)c (�g/ml)d mol/mol)e

10.0�2.53.2�1.3PC (egg yolk)a 2.63�0.622.7�6.3 4.54�1.3PI (DPPI)b 18.4�5.1

4.93�1.424.6�6.8PI (bovine 22.2�6.2liver)b

PI-4-P (bovine 13.22�2.366.1�11.4 66.4�11.4brain)b

135.5�16.0PI-4,5-P2 (bovine 24.67�2.9123.4�14.6brain)b

243.6�29.0PI-3,4,5-P3 272.6�32.4 48.73�5.8(SAPIP3)b

a Positive ions.b Negative ions.c The values given represent the lipid amount that is required on the sample plate to achieve a signal-to-noise-ratio of 5, when

equal volumes of lipid stock solution and matrix solution (0.5 M DHB in methanol with 0.1 vol.% TFA) are applied (1 �l each).The limits were estimated from the lipid concentration dependence of the signal-to-noise ratio.

d Calculated from the data of the preceding column using the respective molecular weight of the lipide Calculated using 1 �l lipid stock solution and 1 �l of 0.5 M DHB matrix solution.


Fig. 5. Signal-to-noise ratio as a function of the PIP2 amounton the plate for MALDI-TOF negative ion mass spectra ofmixtures of PIP2 with egg-PC: PIP2 alone (�), and PIP2 mixedwith 132 (�), 658 (�), or 1315 pmol PC (�). Lipid mixtureswere prepared by mixing appropriate amounts of organic lipidstock solutions. All other conditions were as in Figs. 3 and 4.

at 0.1 vol.% TFA. Higher amounts of TFA de-stroy the homogeneity of the matrix-sample mix-ture and therefore reduce the PIP2 detectability.At 0.5 vol.%, the PIP2 detection limit is higherthan 1000 pmol.

Some authors reported better results forMALDI-TOF MS with samples that tend to forminhomogeneous matrix-sample mixtures, when thesample and the matrix solution are premixed be-fore the application to the sample plate. However,in our case the detection limit for PIP2 was onlyslightly reduced to 104.4�14.4 pmol by thisprocedure.

4. Discussion

In this paper, we reported a detailed study onthe MALDI-TOF mass spectrometric analysis of(poly-)phosphoinositides and lipid mixtures con-taining phosphoinositides.

MALDI-TOF MS is a very convenient andefficient method for the analysis of lipids due tothe simple handling, the fast measurement and thelow substance requirements. Therefore, theused for the calculation of S/N. In the case of

Ca2+, the strong hygroscopic properties of CaCl2did not allow to maintain the necessary vacuum inthe ion chamber of the device. This results in poorspectra quality.

The addition of EDTA also does not lead to animproved PIP2 detection (detection limit: 118.1�19.3 pmol). In general, the homogeneity of thesample-matrix mixture is considerably reduced byaddition of any of the described substances andthe spectra quality is diminished, accordingly.

The acidification of the sample-matrix mixturecould be a conceivable strategy for the improveddetection of charged molecules. For that purposeTFA, that is one of the strongest known organicacids, was added to the DHB matrix solution at aconcentration of 0.1 vol.% for all measurementsdescribed above. Without TFA the detection ofPIP2 is not possible up to the highest testedconcentration (956 pmol). An increase of the TFAcontent to values higher than 0.1 vol.% improvesthe detectability of PIP2. There is an optimum at0.4 vol.% TFA. Here, the detection limit for PIP2

is 69.8�13 pmol in contrast to 123.4�14.6 pmol

Fig. 6. Signal-to-noise ratio as a function of the PIP3 amounton the plate for MALDI-TOF negative ion mass spectra ofstearoyl-arachidonoyl-PIP3 (�) and SAPIP3 in mixture with132 (�), or 658 pmol (�) PC, or in a mixture with 658 pmolPC and 95.6 pmol PIP2 (). All conditions were as in Fig. 5.


Table 2Effect of the sample preparation conditions on the detectableamount of PIP2

Final Detection limit forPIP2 (pmol)concentration

Standard 123.4�14.6preparationtechniquea

Additi�eb

Not detectable1 mMCa2+

(�1000 pmol)311.3�29.2Na+ 1 mM

Na+ Saturated 513.3�32.4organicsolution

Spermine4+ 89.6�17.21 mM118.1�19.31 mMEDTA

0.2 vol.% 110.6�13.2TFA-content of theDHB solutionc

69.8�13.00.4 vol.%0.5 vol.% Not detectable

(�1000 pmol)

Premix of analyte 104.4�14.4and matrixsolutiond

a Described in Section 2.b The addition of cations or chelators to the lipid sample

was done by preparing the stock solution of PIP2 in a mixtureof chloroform, methanol and 1 M HCl (2:1:0.02, v/v/v), thatcontained the corresponding salt at the given concentration.

c The increase of the TFA content of the DHB matrixsolution was done by addition of the appropriate amount ofconcentrated TFA to the methanol solution of DHB.

d The premix of analyte solution and matrix solution wasdone in small glass vials at the ratio 1:1 (v/v).

In general, MALDI-TOF MS spectra cannot beanalyzed quantitatively, if only the measured sig-nal intensity is taken into account. The signalintensity is influenced by factors that cannot beeffectively standardized, e.g. differences in samplepreparation, inhomogeneities of the sample-ma-trix mixture, fluctuations of the laser intensity aswell as different ionization and desorption efficacyfor different molecules. Different approaches canbe used to overcome the limitation of quantitativeinformation from MALDI-TOF MS. For exam-ple, internal standards of the same chemical na-ture as the substance of interest can be used(Duncan et al., 1993; Benard et al., 1999). Afurther approach is to estimate the relative con-centrations of the individual components in amixture from the respective relative peak intensi-ties (Petkovic et al., 2001).

In this paper, the signal-to-noise ratio was in-troduced as a quantitative measure. This attemptdoes not require any addition of other substancesto the sample. It can be done semi-automaticallyusing the standard software packages availablefor commercial MALDI-TOF mass spectrometer.The calculated S/N ratio increases continuouslyover a wide concentration range with the sampleconcentration and, additionally, provides an exactcriterion for the estimation of the detection limit.The value S/N=5 was taken as the detectionthreshold.

The most abundant cellular lipid classes likephosphatidylcholines and phosphatidylethanol-amines are usually measured in the positive ionmode, and exhibit extremely low-intense peaks inthe negative ion mode. On the other hand, due totheir negative charges, phosphoinositides exhibitonly very small signals in the positive ion modeand are much better detectable as negative ions.

Unfortunately, the detection limits for PPI arehigher than for other phospholipids. The detec-tion limit increases with the PPI phosphorylationstate. Both, the higher molecular weight and thehigher negative charge are responsible for thelower PPI detectability with increasing phospho-rylation. It was reported for triacylglycerols thatthe relative signal intensity in MALDI-TOF spec-tra clearly decreases with the molecular weight

method seems to be promising for the investiga-tion of lipids, which are less abundant in cellularsystems as the (poly-)phosphoinositides, or for themonitoring of the turnover of these lipids. Wehave shown that PPI could be analyzed byMALDI-TOF MS, but their detectability de-creases drastically with increasing number ofcharges at the inositol ring and in the presence ofother abundant lipids like phosphatidylcholines.Limits for the detection of these substances byMALDI-TOF MS and some strategies to improvethe detectability of (poly-)phosphoinositides canbe stated explicitly as a result of ourinvestigations.


(Asbury et al., 1999). The detectability of poly-mers and proteins also depends on the molecularweight. It is much easier to detect smaller thanlarger molecules (Hillenkamp et al., 1991). Fur-thermore, MALDI-TOF MS is more successfulwith uncharged than with charged molecules(Stahl et al., 1991). For example, one easily de-tects dextran with a molecular weight of 4000 Dain moderate concentration using a DHB matrix,whereas the detection of the charged dextran sul-fate at comparable concentrations is only possiblefor a molecular weight of up to approximately1000 Da (Schiller and Arnold, 2000). Ananalogous behavior was already reported forsmaller degradation products of cartilage. In thatcase, smaller moderately charged molecules weremuch easier detectable than larger, highly chargedones (Schiller et al., 1999b). Polarity effects canalso contribute to the PPI detection limit. Themore polar a substance, the more difficult it is todetect it by MALDI-TOF MS (Hillenkamp et al.,1991). Phosphoinositides are more polar thanother phospholipids that are well-detectable byMALDI-TOF MS (e.g. phosphatidylcholines) Ad-ditionally, the polarity of the phosphoinositidesincreases with the increasing number of phosphategroups. This can contribute to the increasing de-tection limit with increasing phosphorylationlevel.

One could also imagine that PPI tend to formhigher charged molecular ions, which would occurat the half or a third of the expected molecularweight. It might be that the intensities of themolecular ion peaks are decreased by this effect.However, our studies did not give any evidence ofsuch a behavior. This is in good agreement with aprevious paper showing that the formation ofsingly charged ions is highly favored (Karas et al.,2000).

The detection of PPI is further complicated, ifPPI are analyzed in a mixture with other phos-pholipids. The presence of a substance that iseasily detectable in a mixture may alter the MSresponse of other components in the mixture(Petkovic et al., 2001). Phosphatidylcholine ex-hibits extremely high signal-to-noise ratios even atlow concentrations due to its quaternary ammo-nia group. This group exhibits a preformed posi-

tive charge. This leads to very efficient ionizationand desorption resulting in the sensitive detectionof this lipid species. The same holds for otherlipids, which contain this group, e.g. sphin-gomyelins (Schiller et al., 2000). Consequently,the detectability of individual lipid species in amixture is reduced if PC or sphingomyelins arepresent. In accordance with that, we found thatthe detection limits for PPI are increased drasti-cally in a mixture with PC.

Different approaches to reduce the detectionlimits for PPI were attempted in the present study.It is known that the homogeneity of the matrix/analyte mixture is crucial for the quality ofMALDI-TOF MS spectra. It can be controlled bythe sample preparation procedure. Some authorssuggest the mixing of analyte with matrix prior tothe application to the sample plate (Benard et al.,1999). Usually, however, the homogeneity of sam-ples prepared from PPI and DHB/0.1 vol.% TFAas matrix is very high without premixing. Thus,the premix of matrix and analyte does not im-prove the sample homogeneity to a high extent.Therefore, it does not improve the PPI detectabil-ity in comparison to simply mixing matrix andanalyte on the sample plate.

As clearly shown, the PPI charge state has aparamount influence on the detection limit. Tomodulate the charge state, we varied the cationcontent and the TFA content of the samples.Unfortunately all ionic additives (Na+, Ca2+,Spermine4+, H+ via the TFA concentration) de-creased the homogeneity of the matrix-samplemixture at least at higher concentrations. There-fore, the overall quality of the MALDI-TOF MSspectra is reduced. This effect obviously overcom-pensates a possible improvement of the PPI de-tectability and, consequently, the PPI detectionlimits are only slightly changed.

Although the detailed mechanism of the ionformation in MALDI-TOF MS still remains un-clear (Zenobi and Knochenmuss, 1998), it isknown that its efficiency clearly depends on thechemical nature of the matrix (Fitzgerald et al.,1993). The application of matrices that were de-scribed to be useful for charged compounds(THA; Harvey, 1999) or saccharides (arabinoseosazone; Chen et al., 1997) did not reduce the PPI


detection limits. The latter matrix did not allowdetecting phospholipids at all in our hands (datanot shown). Also the application of 2-hydroxy-5-methoxybenzoic acid was not successful for thedetection of PPI (data not shown). Therefore, ourand other authors experiences that DHB worksbest for the detection of phospholipids (Marto etal., 1995; Schiller and Arnold, 2000) areconfirmed.

Concluding our results, we can describe andexplain the existence of distinct and relatively highdetection levels for PPI by three points: (i) thedetectability of inositol lipids decreases with theirmolecular weight; (ii) it is also decreased by thecharge state; (iii) the analysis of phosphoinositidesin lipid mixtures is restricted by the presence ofeasily detectable lipids, especiallyphosphatidylcholines.

To illustrate the consequences of our findingsfor the application of MALDI-TOF MS as aroutine tool in the lipid analysis of cells or modelsystems, we have to recall that the estimateddetection limits in the range of 50–500 pmol lipidon the plate correspond to lipid stock concentra-tions of about 50–600 �g/ml. This is clearly out-side the concentration range available inbiochemical experiments with PPI. For example,our measurement using PIP3 in a mixture withPIP2 and PC reflects the situation in a conversionexperiment, where PI-3-kinase is incubated withphospholipid vesicles, containing PI-4,5-P2 as theenzyme substrate and PC as vesicle matrix lipid.The PI-3,4,5-P3 generated by the PI-3-kinase isdetected by MALDI-TOF MS only in concentra-tions exceeding a detection limit of about 700pmol. In biochemical experiments this is typicallynot the case. Also the improvements of the PPIdetection by the described changes in the samplepreparation procedure are not as marked asneeded for the detection of these lipids in the lowpicomolar concentration range. Therefore, it isadvisable to pre-separate and concentrate thephosphoinositides from biological lipid mixturesbefore analyzing them by MALDI-TOF MS. Thiscan be done more easily using the second ‘soft’MS ionization technique ESI, since ESI-MS canbe interfaced to liquid chromatography.

Acknowledgements

This work was supported by the DeutscheForschungsgemeinschaft (project AR-283/4, Son-derforschungsbereich 197 ‘Bio- und Modellmem-branen’ projects A9 and A10,Sonderforschungsbereich 294 ‘Molekule in Wech-selwirkung mit Grenzflachen’, and Innovation-skolleg INK 23/A1-1 ‘Chemisches Signal undBiologische Antwort’). The helpful advice of Dr.Haferburg (Faculty for Biosciences, Pharmacyand Psychology of the University of Leipzig) isalso gratefully acknowledged.

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Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)