Paper on methodology

-D-Glucopyranosyl-, D-alanyl- and L-lysylcardiolipin from gram-positive bacteria: analysis by fast atom bombardment mass spectrometry

Correspondence to: W..

	ABSTRACT

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Cardiolipin species substituted on O2 of the middle glycerol moiety with -D-glucopyranosyl, D-alanyl and L-lysyl residues were isolated from different gram-positive bacteria. There respective structures were elucidated by positive and negative mode fast atom bombardment mass spectrometry. The structural heterogeneity due to different fatty acid combinations was documented by up to seven molecular ions. General structural features were derived from diagnostic fragment ions, generated by single cleavage at the phosphodiester moieties in both positive and negative ion mode. A diagnostically important fragment ion for D-alanylcardiolipin was observed in the positive ion mode. It arose from double cleavage of the phosphodiester moieties yielding [NaH(Na) PO₄-CH₂·CH(OCO · CHNH₂ · CH₃) CH₂⁺]⁺. The fatty acid combinations in the phosphatidyl and diacylglycerol ions make it possible to recognize whether saturated and unsaturated fatty acids were selectively or randomly distributed on the two positions of the glycerol moieties. Molecular structures of cardiolipins, derived from mass spectrometric experiments, are in full agreement with those, elucidated by classical chemical analyses.—Peter-Katalinic, J., and W. Fischer. -D-Glucopyranosyl-, D-alanyl- and L-lysylcardiolipin from gram-positive bacteria: analysis by fast atom bombardment mass spectrometry. J. Lipid. Res. 1998. 39: 2286–2292.

Supplementary key words: cardiolipin, D -alanylcardiolipin, D -glucopyranosylcardiolipin, L-lysylcardiolipin, listeria, streptococci (group B), vagococci, FAB MS

	INTRODUCTION

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Phosphatidylglycerol (PtdGro) and bisphosphatidylglycerol (Ptd₂Gro, cardiolipin) are wide-spread negatively charged lipid components in the cytoplasmic membranes of gram-positive and gram-negative bacteria (see reviews by O'Leary and Wilkinson (1) and Wilkinson (2)). Since the discovery of alanylphosphatidylglycerol in Clostridium welchii by MacFarlane in 1962 (3), aminoacylester of PtdGro have been detected in many gram-positive (1) (4) (5), but apparently not in gram-negative bacteria (2). Frequently observed ligands were D-alanine and L-lysine. Glycosylated PtdGro has so far only been found in certain strains of Bacillus megaterium in the form of 2-amino-2-deoxy-ß-D-glucopyranosyl derivatives (6) (7). Later on, a substituted cardiolipin was detected in several strains of Streptococcus group B and identified as -D-glucopyranosylPtd₂Gro (8). In group B Streptococcus, it amounts to approximately 18% of the lipid phosphorus and was as a minor component found in Vagococcus fluvialis (9). From V. fluvialis, D-alanylPtd₂Gro was isolated and characterized. It was shown to occur in all strains tested and contributed up to 38% of the lipid phosphorus (9). L-LysylPtd₂Gro, another cardiolipin derivative, occurs in listeria and was detected in each of the four species tested: Listeria monocytogenes, Listeria innocua, Listeria seeligeri, and Listeria welshimeri. It contributes between 11 and 26 mol percent to the polar lipids (W. Fischer and K. Leopold, unpublished data).

In the present report we describe negative and positive ion fast atom bombardment mass spectrometry (FAB MS) of the three substituted cardiolipins. The fragmentation pattern of phospholipids has been established by previous workers using phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol (10) (11) (12) (13) (14). Cardiolipin was studied by negative ion MS/MS. The sample used contained only one fatty acid species (18:2) and accordingly showed a single molecular ion (15).

	MATERIALS AND METHODS

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Lipids
D-GlcPtd₂Gro was isolated from Streptococcus group B type Ia, Kiel 090 (8), D-AlaPtd₂Gro from V. fluvialis, NCDO 2497 (9), and L-LysPtd₂Gro from L. welshimeri, SLCC 5334 (W. Fischer and K . Leopold, unpublished data). Briefly, lipids were extracted from mechanically disintegrated bacteria by a modified acidic Bligh Dyer procedure, fractionated by column chromatography on DEAE cellulose, and finally individual lipids were purified by chromatography on Iatrobeads (Iatron Laboratories, Tokyo) and/or preparative TLC. As far as possible, the pH was kept at 4.7 in order to avoid base-catalyzed hydrolysis of the labile O-aminoacyl ester bonds (16). The structures of the cardiolipin derivatives, including the characterization of the fatty acid composition, were established by chemical and enzymatic procedures (8, 9; W. Fischer and K . Leopold, unpublished data). The purified lipids were stored at -20°C in CHCl₃–MeOH 2:1 (by vol; slightly acidified with acetic acid).

Fast atom bombardment mass spectrometry
FAB MS was performed on a ZAB-HF mass spectrometer (VG Analytical, Manchester, UK) at the University of Bonn essentially as described (17). The lipids were dissolved in CHCl₃–MeOH 2:1 (by vol) and desorbed from the matrix thioglycerol (positive ion mode) or triethanolamine (negative ion mode) with xenon as bombarding gas. The mass spectra were acquired as single scans in the upscan mode on a AMD DP10 data system, fitted with SAMII (KWS) hardware and SUSY software (AMD Intectra, Beckeln, Germany). Mass values were obtained after calibration with CsI and represent nominal mass numbers.

	RESULTS AND DISCUSSION

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-D-Glucopyranosylcardiolipin (GlcPtd₂Gro), L-alanylcardiolipin (AlaPtd₂Gro), L-lysylcardiolipin (LysPtd₂Gro), and the cardiolipin (Ptd₂Gro) coextracted with the LysPtd₂Gro were analyzed by negative and positive ion FAB mass spectrometry. Molecular ions and fragment ions are summarized in Table 1 and Table 2, the underlying fragmentations are shown in Figure 1 and Figure 2. The constituent fatty acids were identified in the negative ion mode as carboxylate ions (f ', f ) and are listed in Table 1 together with their relative abundance which was determined by GLC (9, 18; W. Fischer, and K. Leopold, unpublished data). The ions g, h, which are observed at m/z 97 and 79 in negative ion mode of all samples (Table 1), belong to the double cleavage ions (Figure 1) and are indicative of the phosphate groups.

Molecular ions
Negative and positive ion FAB mass spectra of GlcPtd₂Gro, AlaPtd₂Gro, and LysPtd₂Gro are shown in Figure 1, 2, 3, and 4. All lipids were composed of several molecular species due to various fatty acids combinations. The peaks of the molecular ions were of higher intensity in negative ion mode and the molecular weights could be derived from them directly. For instance, AlaPtd₂Gro displayed in negative ion mode three molecular ions [M + H⁺]^- at m/z 1474, 1448, and 1446 (Table 1, Figure 2A); in positive ion mode only the molecular ion [M - H⁺]⁺ of the most abundant species appeared at m/z 1476. It was accompanied by [M + Na⁺]⁺ at m/z 1498 (not shown) and, after addition of sodium acetate to the matrix, two species were recognized as [M - 2 H⁺ + 3 Na⁺]⁺ at m/z 1542 and 1514 (Table 2, Figure 2B). Likewise, in positive ion mode GlcPtd₂Gro provided trisodiated molecular ions [M - 2 H⁺ + 3 Na⁺]⁺ at m/z 1661, 1633, 1605 (Table 2). In negative ion mode four molecular ions [M - H⁺]^- were detected at m/z 1593, 1565, 1537, 1511, which were accompanied by a series [M - 2 H⁺ + Na⁺]^- of lower intensity at m/z 1615, 1587, 1559 (not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1. FAB-mass spectrum of D-glucopyranosyl cardiolipin from Streptococcus B in negative (A) and positive ion mode (B).

View larger version (27K): [in this window] [in a new window]   Figure 2. FAB-mass spectrum of D-alanylcardiolipin from Vagococcus fluvialis in negative (A) and positive ion mode (B).

Ptd₂Gro and LysPtd₂Gro, isolated from the same lipid extract of L. welshimeri, possessed a very similar fatty acid composition. This is documented by identical carboxylate ions and the relative abundance of the constituent fatty acids, both shown in Table 1. In negative ion mode the two lipids showed five molecular ions [M - H⁺]^- that differed from each other by 128 amu (Table 1), consistent with the chemically established lysyl substituent (W. Fischer, and K. Leopold, unpublished data).

In positive ion FAB MS, the molecular ions [M + H⁺]⁺ of LysPtd₂Gro were accompanied by a set of ions showing an increment of 92 amu (Figure 4B) and the fragment ions i, i' and n also displayed analogs with this increment (Table 2). A covalently linked constituent, which might have been overlooked on chemical analysis, is unlikely because no adequate molecular and fragment ions were found in negative FAB MS. A complexation with glycerol (molecular weight 92) can also be excluded because glycerol was not added to the analyte/matrix mixture. As the other substituted cardiolipins did not show this feature, one might suspect that the increment of 92 amu was accomplished by complexation with a degradation product of LysPtd₂Gro. An intermolecular transfer of choline and its degradation products (loss of H₂O and loss of H₂O + CH₂) was reported previously to occur in positive ion FAB MS of phosphatidylcholine (11).

View larger version (19K): [in this window] [in a new window]   Figure 3. FAB-mass spectrum of cardiolipin from Listeria welshimeri in negative ion mode.

View larger version (30K): [in this window] [in a new window]   Figure 4. FAB-mass spectrum of L-lysylcardiolipin from Listeria welshimeri in negative (A) and positive ion mode (B).

Fragment ions
The fragmentations, observed in negative and positive ion mode, are summarized in Figure 1 and Figure 2. Fragment a [M - H⁺ - fatty acyl chain]^- and fragment b [M - ⁺CH₂ · CH(OCOR) · CH₂OCOR]^- were observed in the negative ion FAB MS of GlcPtd₂Gro and Ptd₂Gro (Table 1). In positive ion mode, analogous fragments i, i' [M + 2 H⁺ - fatty acyl chain]⁺ and k [M + 2 H⁺ - ⁺CH₂ · CH(OCOR) · CH₂OCOR]⁺ were formed from LysPtd₂ Gro.

In negative ion mode, the diagnostically important ions c are created by cleavage between the oxygen of the phosphatidyl residue and the CH₂ group of the middle glycerol moiety (Figure 1). Phosphatidyl ions c were released from all four compounds (Table 1). From fragment c, two kinds of daughter ions were derived (Table 1): ion d arose by loss of a single acyl residue; ion e by release of a neutral fatty acid molecule (Figure 1).

As shown in Figure 2, phosphatidyl residues were also released in positive ion mode from GlcPtd₂Gro and AlaPtd₂Gro, but the resulting ions l at m/z 691, 719 were upshifted by 46 amu due to the addition of 2 Na⁺ (Table 2). Loss of a neutral fatty acid molecule from l produced the daughter ions m at m/z 437, 463. In addition to phosphatidyl ions l, in positive ion mode diacylglycerol ions n of m/z 577 and 549 were generated. Subsequent loss of a single fatty acyl chain (16:0) led to ions o at m/z 361 and 339 (Figure 2, Table 2).

From the positive ion FAB mass spectrum of Lys Ptd₂Gro, phosphatidyl ions l were absent (Figure 3BDiacylglycerol ions n appeared at m/z 551, 537, and 523, and ions o of m/z 327 and 299 were derived from them by loss of a single fatty acyl chain.

Ions, diagnostic for the substituents
The positive ion mode proved diagnostically particularly important. Double cleavage released the internal phosphoglycerol to which the respective substituent may remain attached (Figure 2).

The most abundant ion of AlaPtd₂Gro was ion q (Figure 2B) which proves the substitution of the middle glycerol moiety with alanine:

P O CH₂ CH O CO CH CH₃OOH(Na)q: m/z 248 (270)NaOCH₂⁺NH₂

In the case of GlcPtd₂Gro, the situation is less clear. The glucosylglyceromonosodium phosphate (ion p) provides an ion at m/z 339, the disodium form an ion at m/z 361.

P O CH₂ CH O C₆H₁₁O₅OOH(Na)p: m/z 339 (361)NaOCH₂⁺

As shown in Table 2, the fragments at m/z 339 and 361 also correspond to the monoacylglycerol ion o and its monosodium form, containing octadecenoic acid.

In the positive ion FAB MS of LysPtd₂Gro an ion of m/z 305 corresponding to the monosodiated lysylglycerophosphate ion was not present. The identity of the ion at m/z 327 as its disodiated form (Figure 3B) is not unambigous because ion o, resulting from loss of a single acyl chain from fragment n, has the same mass/charge ratio (Table 2).

Ions, diagnostic for fatty acid distribution
The phosphatidyl ions c, observed in negative ion mode, provide evidence for the fatty acid distribution on the two positions of the glycerol moieties. GlcPtd₂Gro yielded two species at m/z 673 and 645, each containing one saturated and one unsaturated fatty acid. This is in accord with earlier results of enzymatically performed fatty acid distribution analysis which showed hexadecanoate and octadecanoate preferentially linked to O1 of the glycerol moiety, hexadecenoate and octadecenoate to O2 (18). By contrast, the phosphatidyl ions c of AlaPtd₂Gro comprised five species at m/z 699, 673, 671, 645, and 643, with substantial abundance of the species at m/z 671 (Figure 2B). Only the ions at m/z 673 and 645 contained the combination of saturated and unsaturated fatty acid, in the other species two unsaturated fatty acids were combined (Table 1). In positive ion mode, species containing two unsaturated fatty acids were represented by the ions of m/z 717 and 689 (Table 2). These data indicate, that AlaPtd₂Gro, in contrast to GlcPtd₂Gro, displays a more random distribution of fatty acids.

	FOOTNOTES

¹ Present address: Inst. Medizinische Physik und Biophysik, Universität Münster, Germany.

	ACKNOWLEDGMENTS

The work in the laboratory of W.F. was supported by the Deutsche Forschungsgemeinschaft (grant Fi 218/7-1). We thank Professor H. Egge, University of Bonn, for the use of the mass-spectrometer.

Manuscript received April 1, 1998; and in revised form July 6, 1998.

Abbreviations: PtdGro, Phosphatidylglycerol; Ptd₂Gro, cardiolipin, bisphosphatidylglycerol; AlaPtd₂Gro, D-alanylcardiolipin; GlcPtd₂Gro, D-glucosylcardiolipin; LysPtd₂Gro, L-lysylcardiolipin; FAB-MS, fast atom bombardment-mass spectrometry

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