RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9, 1109-1 114 (1995)

Quantification of Polyphosphoinositides Using Selected Ion Monitoring Electrospray Mass Spectrometry Peter Michelsen,* Nycomed Innovation AB, S-205 12 Malmo, Sweden

Bengt Jergil Biochemistry, Chemical Center, Lund University, S-22100 Lund, Sweden

Goran Odham Chemical Ecology-Ecotoxicology, Lund University, S-223 62 Lund, Sweden

SPONSOR REFEREE: Professor David E. Games, University of Wales Swansea, Swansea SA2 8PP, UK

Polyphosphoinositides (PIP) and (PIP,) show prominent negative singly and doubly charged deprotonated molecules in electrospray mass spectrometry. These ions can be used for quantification of PIP and PIP, in the low picomole range, without prior chromatographic separation, using selected ion monitoring and consecutive measurements of the signals from the deprotonated singly charged molecules. The dose response curves for both compounds are linear. In a complex matrix consisting of polar lipids (Folch extract) PIP and PIP, monitored at mlz 965.4 and 1045.5 (stearoyl and arachidonoyl) were determined in the low picomole range, at a flow rate of 100 pL/min. Collision-induced decomposition of PIP and PIP, using a mixture of xenon and argon at 25 eV afforded identical high mass ions formed by loss of a molecule of water from PIP and a phosphate group and a molecule of water from PIP,. The results indicate that polyphosphoinositides, and biologically relevant changes in their concentrations, can be quantified directly in cells and cellular membranes by selected-ion monitoring with electrospray mass spectrometry.

Phospholipids are the major structural components forming the lipid bilayer of membranes. In addition to their structural role, some phospholipids are key ele- ments in cellular signalling systems,'-2 thereby undergo- ing rapid concentration changes in response to physio- logical stimuli. In particular, the metabolism of phosphoinositides is coupled to the action of numerous hormones, transmitter substances and growth factors as part of signal-transducing mechanisms across the plasma membrane. There is also evidence that phosphoinositides are involved in regulatory events within the cell, as they have been identified in the cytoskeleton3 and the cell n u c l e u ~ . ~

Phosphatidylinositol (PI) may undergo enzymatic phosphorylation in the 3- or 4-position of the inositol ring,' and the PI phosphates (PIP), in turn, may be further phosphorylated. The PI 3-phosphate series is much less abundant than the PI 4-phosphate one, and appears to be coupled to the action of growth factors.6 PI 4-phosphate can be phosphorylated in the 5-position to phosphatidyl inositol 4,5-biphosphate (PIP,), which is crucial for hormone and transmitter signalling across the plasma membrane. Thus, PIP, is cleaved by phos- phoinositidase C to diacylglycerol and insositol 1,4,5- triphosphate in an agonist-triggered event, with the cleavage products acting as intracellular signal sub- stances eliciting the proper physiological response.

A sensitive and convenient analysis method to follow variations in the phosphoinositides would facilitate stu- dies of hormone and transmitter action.

Phosphoinositides may be analyzed following radio-

* Author for correspondence.

labelling of the experimental material, but this method does not allow an easy quantification unless the specific labelling is known. Furthermore, labelling to equilib- rium may be difficult to achieve as there may be more than one phosphoinositide pool in cells,' having differ- ent turnover rates. Alternatively, phosphoinositides may be quantified chemically by phosphate ana ly~is ,~ but this method is rather insensitive (nanomole range). In both instances the phosphoinositides must be separ- ated prior to analysis. This can be done either by thin- layer-chromatography (TLC)' or by high performance liquid chromatography (HPLC),9 in the latter case preferably after deacylation to facilitate separation of the polyphosphoinositides."'

The literature is scarce regarding mass spectrometric investigations of polyphosphoinositides. The thermal lability and liability to fragment in the ion source makes very soft ionization methods necessary. Nakamura et al." reported fast atom bombardment (FAB) studies of the 9-anthryl derivatives of natural PIPS isolated with preparative TLC. The mass spectra showed di- and tri- sodiated molecules of the anthryl derivatives.

Quite recently Han and Gross', studied positive- and negative-ion electrospray mass spectrometry for the common human erythrocyte plasma membrane phos- pholipids. Sodiated molecule adducts were observed in the positive-ion mode for phosphatidylethanolamine (PE) and phosphatidylcholine, whereas intense peaks due to deprotonated molecules were observed for PE. The sensitivity was reported to be 2-3 orders of magni- tude better than that in FAB.

The present work was performed to explore in more detail the usefulness of electrospray mass spectrometry for the determination of polyphosphoinositides, and in

CCC 0951-4198/95/121109-06 0 1995 by John Wiley &'L Sons, Ltd

Received 25 August 1995 Accepted 25 August 1995

1110 SIM-ES MS OF POLYPHOSPHOINOSITIDES

particular selected ion monitoring (SIM), possibly com- bined with tandem mass spectrometry, as an alternative to chromatographic separation of PIP and PIP:, and quantification by conventional methods.

EXPERIMENTAL

Isolation of PIP and PIPt

The phosphoinositides were p~r i f i ed '~ from a bovine brain extract (Folch Fraction Type I., Sigma, St Louis, MO, USA).

Mass spectrometry Mass spectrometry was carried out on a VG Quattro I1 mass spectrometer (Fisons Instruments, VG Organic, Altrincham, UK) equipped with a pneumatically assisted electrospray ion source and a VG Mass Lynx data handling system. Negative-ion electrospray recordings were performed. Full scan spectra, from m/z200-1200, were obtained at a scan speed of 250 u/s with unit mass resolution.

The collision-induced decomposition (CID) spectra were recorded at 25 eV collision energy with a mixture of xenon + argon (25 : 75 v/v) as collision gas. Dose

PIP

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Figure 1. Negative-ion electrospray mass spectrum of (a) L-a-phosphatidylinositol 4-monophosphate and (b) L-a- phosphatidylinositol 4,5-biphosphate.

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Figure 2. SIM signals (counts) of four consecutive loop injections of L-a-phosphatidylinositol 4- monophosphate and L-a-phosphatidylinositol 4,5-biphosphate (17 pmol/pL of each compound).

response curves were constructed in SIM at a dwell time of 0.08 s and an interchannel delay of 0.02 s.

Sample introduction Injections of PIP : PIP, mixtures at various concentra- tions were performed using a 10 pL loop. The solvent consisted of a mixture of chloroform + methanol + water (10.20:8: v/v/v), and the flow rate during the quantitative experiments was 100 pL/min. A Varian model 9012 gradient pump (Varian Chromatography Systems, Walnut Creek, CA, USA) served as solvent deliverer.

RESULTS AND DISCUSSION The initial studies comprised the recording of full spectra of PIP and PIP, in both the positive- and negative-ion modes. It became immediately evident that the total current of negative ions was at least one order of magnitude higher than that of positive ions. Furthermore, the positive-ion spectra were poor regarding high intensity molecular related fragments. As a consequence, all further studies were performed on negatively charged ions.

Figure l(a) and (b) show the full negative-ion spectra of PIP and PIP,, respectively, isolated from bovine brain. PIP gives rise to a prominent peak at m/z 965.7 (Fig. l(a)), a value which tallies with that of deproto- nated molecules of PIP containing stearoyl (18 : 0) and arachidonoyl (20 : 4) residues. Interestingly, the base peak is formed from doubly charged molecular-related ions at m / z 482.6. Low intensity ions in the molecular- ion range indicate the presence also of small amounts of other acyl residues. In the negative-ion full spectrum of PIP, (Fig. l(b)), deprotonated molecules form the base peak of m/z 1045.6 indicating the presence of the same two acyl residues as in PIP. The doubly charged ions of m/z 522.5 represent less than half of the intensity of the singly charged ions. As in the case of PIP, small amounts of acyl residues of different chain lengths and unsaturation were observed in the molecular weight range.

As it was considered of considerable future interest to couple, on line, the mass spectrometer to an HPLC system, chromatographic flow rates of 100, 300, and

1000 pL/min, matching capillary, narrow bore, and standard columns were studied for electrospray in the negative-ion mode using PIP,. These flow rates corre- spond to column internal diameters of 1, 2, and 4.6 mm, respectively. In the molecular weight reange the spectra were very similar, but it was noted that the signal-to-noise ratio decreased with increasing flow rate, for instance by a factor of 5 when changing from 100 to 1000 pL/min. Considering the accessibility of commercial columns, narrow bore columns operating at flow rates of about 300 pL/min appear to be a good compromise in future studies. This finding agrees with that for plasmaspray ionization of similar phosphory- lated inositides. l4

A mixture of PIP and PIP2 in equimolar proportions of 17pmol/pL in the solvent mixture described was introduced. The mass spectrometer was adjusted to shift repeatedly between masses of the deprotonated molecules during loop injection, and to measure the signal obtained. The dwell time on each mass was 0.08 s allowing for about 300 measuring points per com- pound. Figure 2 shows the SIM signals of the peaks at mlz 965.5 (PIP) and 1045.6 (PIP,) from four consecu- tive loop injections. The standard deviation (SD) (n=4) of about 2.5% for PIP and 5.8% for PIP2 indicates that the variation in the results obtained was small. Interestingly, the signal for PIP was about nine times higher than that for PIP2 despite the fact that the ions of 1045.6 form the base peak in the mass spectrum (cf. Fig. 1).

Dose-response curves for the two phosphorylated inositides were constructed by measuring the SIM signals at the above masses of the mixture at different concentrations (Fig. 3). Both curves show good linear- ity (? = 0.9960 for PIP and ? = 0.9998 for PIP,), indi- cating straightforward potential for quantification in the low picomole range.

The calibrations were tested by injecting a mixture of PIP and PIP, (picomole range) in a molar proportion of 1 : 3 under mass spectrometric conditions identical to those used for the construction of the dose response curves. The ratio of counts obtained indicated the proportion 24.8 : 75 : 2, which was well within the error of dilution.

Optimal proof with respect to the identity of the analyte may be obtained using CID of the molecular-

1112 SIM-ES MS OF POLYPHOSPHOINOSITIDES

related ions observed in the full spectrum. Hereby, fragments used in SIM are correlated with a high degree of certainty to ions from the parent ions. Figure 4(a) and (b), show daughter ions in CID of PIP and PIP, respectively, at a collision energy of 25 eV using a mixture of xenon and argon as collision gas. The PIP daughter ions of highest mass (mlz 947.0) are formed by loss of molecules of water from the deprotonated negatively charged molecules. For PIP,, under the same collision conditions, the deprotonated molecules unfortunately lose phosphate groups and molecules of water leading to ions of identical mlz to those formed from PIP. In this particular case, it is thus not possible to utilize the inherent capability of tandem mass spec- trometry, using multiple reaction monitoring, to add further proof as to the identity of the molecular related

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ions. It also seemed appropriate to evaluate the benefit of

the proposed technique for the determination of PIP and PIP2 in a natural complex matrix. A mixture of the total polar lipids (30 pgIpL) in a bovine brain extract was therefore subjected, without further purification, to full-scan mass spectrometry. Figure 5 shows the high-mass range negative-ion mass spectrum obtained, where the deprotonated molecules of PI, PIP and PIP, are clearly recognized at mlz 787.9, 965.4, and 1045.3, respectively (acyl residues 18 : 0 and 20 : 4). Three con- secutive measurements of the sample diluted with an equal volume of mobile phase produced signals in SIM at mlz 965.4 and 1045.3 corresponding to a concentra- tion of PIP and PIP2 of 7.4 k 0.2 and 7.2 f 0.2 pmollyl, respectively, (cf. Fig. 4). We have found that the Folch

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0 20 40 60 80 100 120 140 160 Concentration (pmole/pL)

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Figure 3. SIM dose response curve for (a) L-a-phosphatidylinositol 4-monophosphate in the presence of L-a-phosphatidylinositol4,5-bisphosphate and (b) L-a-phosphatidyEnositol4,5-bisphosphate in the presence of L-a-phosphatidylinositol4-monophosphate.

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wr Figure 4. Negative-ion electrospray daughter mass spectrum of (a) mlz 965 of L-a-phosphatidylinositol4-monophosphate and of (b) mlz 1046 of L-a-phosphatidylinositol4,5-bisphosphate. Collision energy 25 eV, collision gas xenon+ argon, 25: 75 (vlv) at 1.5 mbar.

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Figure 5. High-mass range negative-ion mass spectrum of phospholipids in a bovine brain extract.

extract used contains 10-20% of phosphoinositides of which about 1% constitutes PIP and PIP2. The total concentration tallies well with these findings. Furthermore, the results show that PIP and PIP2 are present in nearly equal amounts and that each of the analytes can be quantified also when they constitute only cu. 0.05% of the total phospholipids in a complex mixture.

In conclusion, our results open up the possibility for the rapid quantification of low levels of phospho- inositides, and physiological changes in their concentra- tions, in cells and cellular membranes without prior separation of the lipid extract.

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1114 SIM-ES MS OF POLYPHOSPHOINOSITIDES

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