Fragmentation of lipopolysaccharide anchors in plasma desorption mass spectrometry

Journal of Mk'robiological Methods ! 5 ( ! 9921 ~'5 ! -- ! 66

151

1992 Elsevier Science Publishers B.V. All. rights reserved 016"7,,. , - "70~' ,_~, /0"~' ,,...~ ¢, _, ~.,.,_,~-q MIMET 00481

Fragmentation of lipopolysacchafide anchors in plasma desorption mass spectrometry Rong Wang 1, Ling Chen 1, Robert J. Cotter ~, Nilofer Qureshi 2, and Kuni Takayama 2. 3 ~Middle Atlantic Mass Spectrometry Facility, Department of Pharmacologg' and Molecular Sciences, Johns Hopkins Universit.v School of Medwine, Baltimore, MD 21205, USA, 'Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, Madison, 1~7 53705, USA and 3Department of Bacteriology, University of Wisconsin, Madison, ~.7 53 706, USA (Received 1 July ! 991; revision received 12 November 1991; accepted 21 November 1991)

Summary Plasma desorption mass spectrometry (PD-MS) was employed for the structural analysis of lipid A's derived from the lipopolysaccharides (LPS) of Escherichia coli D31 m4, Rhodopseudomonas sphaeroides ATCC 17023, and Rhodopseudomonas capsulata ATCC 23782. The deep rough chemotype LPS (ReLPS) of E. coli D31m4, a lipid A containing two 2-keto-3-deoxyoctonate (KDO) units, was also analyzed. Several forms were examined, including the mono- and di-phosphoryl lipid A's, and lipid A's methylated at the phosphate group. Positive ion PD-MS gave molecular ions, ions corresponding to the loss of ester-linked fatty acyl and glycosidic phosphate groups, oxonium ions formed by the cleavage of the distal sugar, and ions resulting from the two-bond cleavage of the reducing-end sugar. Negative ion PD-MS gave molecular ions and fragmentation products of the phosphate group and fatty acyl anions in the low mass region. The presence of the KDO group on the lipid A structure had little effect on the fragmentation pattern of the rest of the molecule of ReLPS. PD-MS of lipid A has allowed us to determine the molecular weight, the distribution of the fatty acyl groups in both the distal and reducing-end sugars, the nature of the O-linked fatty acyl groups, and the presence of a glycosidic-linked phosphate. In combination with proton nuclear magnetic resonance spectroscopy, PD-MS allows one to determine the complete structure of lipid A.

Key words: Lipid A; Lipopolysaccharide; Plasma desorption mass spectrometry; Structural analysis

Correspondence to: R.J. Cotter, Middle Atlantic Mass Spectrometry Facility, Department of Pharmacology and Molecular Sciences, Johns Hopkins University 5ciaooi of Medicine, Baltimore, MD 21205, USA Abbreviations: LPS, lipopolysaccharides; MS, mass spectrometry; FAB, fast atom bombardment; LD, laser desorption; PD, plasma desorption; ReLPS, deep rough chemotype LPS; MLA, monophosphoryl lipid A; DLA, diphesphoryl lipid A; KDO, 2-keto-3-deoxyoctonate.

152 Introduction

Lipopolysaccharides (LPS) are amphipathic glycolipids found on the outer surface of the outer membrane of Gram-negative bacteria [1]. LPS has been shown to elicit a wide variety of immunological responses, including B-cell mitogenicity, macrophage activation, and induction of several key cytokines of macrophage-monocyte origin (interferon, colony stimulating factor, tumor necrosis factor, and interleukinI) [2]. LPS are comp.:,~ed of three structural regions: the hydrophilic O-specific polysaccharide region, the common core oligosaccharide region, and the hydrophobic lipid A region which forms the anchor for the LPS bound to the outer membrane by hydrophobic interaction [3]. Lipid A itself is responsible for a number of pathophysiological ~.ffects associated with Gram-negative bacterial infection. These effects include induction of endotoxin shock, pyrogenicity, hemodynamic changes, and disseminating intrava~cular coagulation [4]. The complete structure of lipid A was established in 1983 [5, 6] using a preparation from the LPS of Sahnoneila typhimurium G30/C21 by using positive ion FAB-MS, proton nuclear magnetic resonance spectroscopy, and chemical analyses. The basic structure of lipid A consists of a mono-, di-, or a triphosphor~ Jated ~-l,6linked o-glucosamine disaccharide with five to seven fatty acyl groups attached to the disaccharide through amide linkages (at the 2- and 2'- positions) and ester linkages (at the 3- and 3 '- positions). The structure of the ReLPS (deep rough chemotype) from Escherichia coli and monophosphoryl lipid A (MLA) from the Salmonella strains and E. coli are shown in Fig. 1. We have used fast atom bombardment mass spectrometry (FAB-MS) [5, 7] and infrared laser desorption mass spectrometry (LD-MS) [8, 9] for the analysis of highly purified, intact lipid A's. Each ionization technique has its own instrumental characteristics and distinct advantages_ Althallgh it ha~ ha~n pn~ihl~ tn n h t ~ i n positive ion FAB-mass spectra for the underivatized MLA [5, 10], LD-mass spectra are more easily obtained for lipid A in which the phosphate groups have been converted to their methyl esters. This is not a disadvantage since both dimethyl MLA and tetramethyl DLA are conveniently purified by reverse-phase high performance liquid chromatography according to the number and chain-length of the fatty acyl groups [7]. In addition, LD-MS of the derivatized lipid A's in the presence of ;(C! r~roduces twobond ring cleavages of the MK ÷ ion, which unambiguously establish the identity of the fatty acryl groups at the distal and reducing-eed ,~t~ftars ~':'i In 1974, Torgerson et al. [11] introduced the techn;qae i~.nown as plasma desorption mass spectrometry (PD-MS) utilizing ~he fission fragments from 252Cf as the ionization source and a time-of-flight mass spectrometer for mass analysis. This was further described by Macfarlane and Torgerson in 1976 [12]. Since then, PD-MS has been widely used in studies of macrobiomolecular structures, most notably peptides with molecular weights up to 35 kDa [13]. In the course of our structural studies of complex carbohydrates, glycolipids, and glycopeptides as well as the lipid A's and LPS obtained from Gram-negative bacteria, we have made a number of interesting observations. First, while LD-MS was most effective for the detailed analysis of methylated lipid A, PD-MS was more successful for the analysis of larger structures, in particular, the ReLPS, which contains two additional 2-keto-3-deoxyoctonate

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Chemical structure of (a) R e l P S flora E. coli D31m4 and (b) MLA obtained from the LPS of E. coli arid Sc,tmoneila strains.

(KDO) units (Fig. 1a) [14]. Secondly, some of the two-bond ring cleavages observed in LD-MS, but not in FAB-MS and characteristic of the mass of the distal structural portion are also observed in PD-MS. Since PD-MS also yields an oxonium ion, it appeat's to be able to provide the same structural information as the combined LDand FAB-mass spectral analyses. Finally, PD-mass spectra can be easily obtained from underivatized lipid A. Thus, we began a more systematic study of both the derivatized (methy~ated) and ~nderi,latized (free acid) lipid A obtained from the LPS of several Gram-negative bacteria, using PD-MS.

154 Ma|erials and Methods

Growth of bacteria The procedure for the growth of E. coli D31 m4 and Rhodopseudomonas sphaeroides ATCC 17023 have been previously described [ 1 4 - 17]. Rhodopseudomonas capsulata ATCC 23782 was grown photoheterotrophicaily in medium 550 (ATCC) at 26 °C (12 days), and harvested by using the cell concentrator (Millipore Corp.) and c . ~ n t r~ f i l ~

Preparation o f LPS The procedure for the preparation of LPS from E. coli and R. sphaeroides have been previously described [14, 16, 17]. For the preparation of LPS from R. capsulata, cell paste (700 g) was extracted with 4 1 each of ethanol/n-butanol (3:1, v/v) for 2 h, the same solvent overnight, and acetone for 2 h (twice), to remove the contaminating pigments. Crude cell wall was prel~ared from these acetone-dried cells. The dry cells (50 g) were suspended in 100 ml of potassium phosphate buffer, pH 7.0, passed through a French pressure cell at 15 000 psi, centrifuged at 10 000 x g for 30 min. The residue was washed twice with the buffer, once with water (100 ml each), then lyophilized to yield 14.5 g of crude cell wall. LPS was extracted from the cell wall preparation by the hot phenol-water procedure [18]. From 14.5 g of crude cell wall, 610 mg of LPS was obtained in the phenol layer.

Preparation o f lipid A The preparation of lipid A's from the LPS of E. coli and R. sphaeroides and their purifications have been previously described [15, 16, 17]. The procedure for the preparation of lipid A (MLA and DLA) from the LPS of R. capsulata was identical to that of R. sphaeroides [16, 17]. Purified lipid A's were methylated by treatment with diazomethane, and the methylated lipid A's were further purified and separated by reverse-phase high performance liquid chromatography as previously described [7].

Sample preparation for PD-MS The free acid forms of lipid A were dissolved in chloroform-methanol (1:1, v/v) and the methylated forms of lipid A were dissolved in chloroform-methanol (4:1, v/v). About 1 - 2/~g of sample in 10 #1 of solution was electrosprayed onto a mylar backed aluminum foil to cover a surface area of 1.35 cm 2 and dried.

Mass spectrometry PD-mass spectra were obtained on a BIO-ION Nordic (Uppsala, Sweden), BIN10K time-of-flight mass spectrometer with a 10 ~Ci 252Cf source and a DEC 11/73based data acquisition system. The fission fragment flux through the sample was about 1500/s/cmL An acceleration voltage of 14 kV to 16 kV was used for positive ion spectra, while - 12 kV to - 14 kV was used for negative ion spectra. The time

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Positive (a) and negative (b) ion PD-mass spectra of E. coli MLA.

resolution was 1 ns/channel. Mass assignments were made by determining the timeof-flight (channel) centroid of each peak and comparing these with the centroids of H + and Na + ion (or H - and C N - in negative spectra) appearing in the same spectra. Mass resolution is generally between one part in 300 to 500, while mass measurement accuracy of the resultant unresolved isotopic ion cluster is O. 1°70 to 0.01 070. Results and Discussion

The positive and negative ion PD-mass spectra of underivatized E. coli MLA are shown in Fig. 2. The spectra show characteristic peaks in several regions corresponding to: (a) molecular ion species and in some cases molecular ion dimers; (b) high mass fragment ions which can be used for structural analysis (to be shown later); (c) low mass fragment ions characteristic of specific lipid A moieties (to be shown later); and (d) atomic ions (i.e. H + and Na +) used for mass calibration.

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Fig. 4. Molecular ion regions of PD-mass spectra of E. coli MLA. (a) Positive ion mass spectrum of underivatized MLA, (b) positive ion mass spectrum of methylated MLA, (c) negative ion mass spectrum of underivatized MLA, and (d) negative ioa mass spectrum of methylated MLA.

PD-mass spectra o f lipid A from the LPS of E. coli The E. coli lipid A is almost exclusively hexaacyl [ 15, 19, 20] consisting of O-linked myristoxymyristate at the 3 '-position (R~), N-linked lauroxymyristate at the 2"position (Rz), O-linked 3-hydroxymyristate at the 3-position (R 3) and N-linked 3-hydroxvmvri~tate :at the 2-nn~itinn ~'R .~ (~ee Fig~ 1h and ~ fnr tk/[lA) We e~nminPcl hnth the free acid and methyl ester forms of this lipid A of known structure by PD-MS to determine: (a) their relative ability to form molecular ions under heavy ic-', Mev bombardment, and (b) the kinds of fragment ions produced as structural information. Molecular ion. The formation of a molecular ion is important in determining the molecular weight of lipid A. In general, MLA was expected to be more stable to the PD-mass spectral ionization process than DLA which contains a more labile, glycosidic phosphate. We found however, that all of the E coli lipid A's tested were sufficiently stable to yield molecular ions by PD-MS. The molecular ion regions of the MLA from E. co6 are shown in Fig. 4. In the positive ion mode, both the free acid (Fig. 4a) and methyl derivative of M L A (Fig. 4b) gave intense pseudo-molecular ions (MNa+) at m / z 1741 and 1769, respectively. The free acid also formed a weak protonated molecular ion MH + at m / z 1719 and a species containing two sodium ions (MH - H + 2Na) + at m/z 1763. The methyl derivative formed an (M - 14 + Na) + ion at m / z 1755. This can be interpreted as the loss of a methyl group accompanied by H-transfer and the formation of a pseudo-molecular ion with the attachment of the sodium ion.

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High mass region of the positive ion (a) and negative ion (b) mass spectra of underivatized E. coli MLA.

In thc negative ion mode, the free acid form of MLA gave (M - H)- ion at m/z 1717 arid (M - C H 3 ) - ion at m/z 1703 (Fig. 4c), whereas the methyl derivative gave MCI- ion at m/z 1781 and ( M - C H 3) ion at m/z 1731 (Fig. 4d). The signal to noise ratios (and therefore mass assignment accuracies) of positively charged molecular ion peaks were better than that of the negatively charged molecular ions. The free acid

i58 form of lipid A gave a better molecular ion signal in the negative ion mode (and was less complex) than the methyl derivative.

Major fragments oflipidA. We looked for major fragment ion peaks in the high mass region of the spectrum that would provide structural information on the E. coli lipid A's. The free ac:,d form of MLA was examined in both the positive and negative ion modes and the results are shown in Fig. 5. A major feature of this region is the loss of the ester-linked fatty acyl groups. For example, in the positive ion mass spectrum (Fig. 5a), cleavage of the ester lm~,,tge t,ctwcc, the ~.,u~"-'-oo.y~" group a,,u""aoxygen at .ho,,,. 3 "position resulted in the loss of R~ (minus 437 amu) (refer to Fig. 3) from the MNa + ion (m/z 1741) to form a peak at m/z 1304. Since the formation of an even-electron ion is expected in desorption mass spectra, it is likely that this cleavage is accompanied by H-transfer from or to the sugar moiety, as well as simple cleavage without H-transfer, although these could not be resolved in the spectrum. Similarly, the losses of 243 and 227 amu from MNa ÷ to yield ions at m/z 1498 and 1514, respectively, can be attributed to the loss of R 3 by cleavage at either side of the oxygen atom of the ester. Generally, we have observed that the cleavage of the fatty acyl side of tl,e ester oxygen is more probable than at the oxygen-sugar bond by PD-MS. The peak at m/z 1535 is interpreted as the loss of 184 amu from the MH + ion. This peak corresponds to a loss of lauric acid from R 2 accompanied by H-transfer from the remaining hydroxymyristate group. In the negative ion mass spectrum (Fig. 5b), we noted that anions can be produced by the loss of hydrogen to form an (M - H)- ion at m/z 1717 and a methyl group to form (M - CH3)- ion at m/z 1703. Anions can also be formed by the loss of fatty acyl ester groups. Thus the losses of 227 and 437 amu correspond to the formation of (M - R3)- and (M - R~)- ions at m/z 1490 and 1280, respectively. These processes cannot be distinguished from losses of neutral species from the (M - H ) ion. Such neutral losses do occur. For example, the peaks at m/z 1474 and 1264 represent losses of R30 and RiO respectively, followed by H -transfer to or from the resultant fragment ion. The peak at m/z 1051 results from the loss of both RIO and R30. It should be noted that preferential losses of ester-linked (but not amide-linked) fatty acyl groups provide important structural information as to their location on the disaccharide backbone. Negative ion PD-MS was shown to selectively cleave only the fatty acyl esters linked directly to the sugar whereas positive ion PD-MS was less discriminating. Thus when we observed the loss of 437 and 227 amu from the (M H)- molecular ion, it indicated that these losses occurred on the sugar-linked ester groups of R~ and R 3. The loss of 183 and 211 amu (representing the normal Cl2 and C!4 of the acyloxyacyl groups) are not seen in negative ion PD-MS. In the positive ion PD-MS, an oxonium ion (a peak at m/z 1088) is formed by the cleavage of the glycosidic bond between the distal and reducing-end sugars [5] (Fig. 5a). The origin of the peak at m/z 1188 is not known. The difference between the masses of the oxonium ion and molecular ion reveals the fatty acid distribution in the two sugars. The conclusion based solely on the results of negative ion PD-MS and the formation of oxonium ion in positive ion PD-MS is that R l is C!4OC14(437 amu) and that R 3 is OHCI4 (227 amu). It then follows that R 2 is Cl2OCl4and R 4 is OHCI4 (Fig. 3). 1"--1.~

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Low mass fragmentation. The low mass region of the negative ion PD-mass spectra

reveals peaks corresponding to fatty acid and phosphate anions. In this mass range, high precision mass measurements could be obtained. The ester bonds were cleaved to yield the carboxylate anions R ! 'O- (m/z 227), R 2'O- (m/z 199), and R30-

160

(m/z 243) (Fig. 6a), where RI 'and Rz'are fatty acyl groups esterified to the hydroxy fatty acids. Fragment ions at m/z 79 and 97 corresponds to the phosphate anions PO~- and H : P Q - , probably formed by cleavages at two different sites. The degree of methylation of the nonreducing end phosphate group of dimethyl MLA can be determined by negative ion PD-MS (Fig. 6b). The spectrum showed the presence of the (CH3)HPO 4- and (CH3)2PO 4- anions at m/z 111 and 125, respectively. In the tetramethyl DLA, it was possible to determine whether it contained two monophosphates or a single pyrophosphate group by the presence of the methylated phosphate anions at m/z 111 and 125 and the absence of the (CH3)3P207- anion at m/z 219 (Fig. 6c). Although the low mass region ( 1 0 - 100 amu) of the positive ion PD-mass spectrum (data not presented) showed fragmentation characteristics of alkyl groups, less structural information was available as compared to negative ion ~'D-mass spectrum. The effect o f methylation on the fragmentation o f lipid A The intensity of the molecular ion peaks of lipid A in both free acid and methylated forms are about equal. However, the intensity of the fragment ion peaks are slightly lower for the methylated lipid A. The partial PD-mass spectrum of dimethyl MLA (HPLC fraction A) from the LPS of R. sphaeroides is shown in Fig. 7. Although the fatty acyl composition of the three lipid A components is given in Table 1, only

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!6! TABLE 1 FATTY ACYL DISTRIBUTION OF R. SPHAEROIDES LIPID A

Mr, di-

HPLC fractions

Fatty acyi distribution

methyl MLA

A B C

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Ri

R2

R3

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OHCIo OHCI ° OHClo

keto-CI4 keto_C14 OHC14

Refer to Fig. 3 for the general structure of MLA. The dimethyl MLA obtained from the LPS of R. sphaeroides was fractionated by HPLC on a Cl8-bonded silica cartridge as previously described and the resulting three major fractions were recovered and analyzed by FAB-, LD-, and PD-MS [16, 17]. OHCi0, hydroxycaproate; OHCI4, hydroxymyristate; keto-Ci4, oxomyristate; A7-C14OC14, A7-tetradecenoyloxy myristate; C14OC14, myristoxymyristate.

fraction A will be discussed. The spectrum shows abundant molecular ions (MNa + at m/z 1470) and very weak fragment ions resulting from the loss of O-linked fatty acyl groups and cleavage of the glycosidic bond. The peaks at m/z 1299 represents loss of a hydroxy capryl group (either R 1 or R3) by cleavage of the carbonyl-oxygen bond without H-transfer (MNa ÷ - 171). The peak at m/z 1282 corresponds to the

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1~.-

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same loss except that the neutral fatty acid is released to yield MNa + - 188. The peak at m/z 1243 is due to the loss of the myristoyl moiety of the acyloxyacyl group, R 2, to yield MNa* - 227. This cleavage occurs between the ester oxygen and hydroxymyristoyl moiety. An oxonium ion peak was observed at m / z 875 as well as a peak corresponding to the two-bond cleavage of the reducing-end sugar to yield a distal ion fragment at m/z 957. Cleavages of the phosphate ester or amide bonds were not observed. The low intensity of the fragment ions might be due to changes in conformation and electron distribution caused by methyl groups on the phosphate. This could make the "" " " acyl ester '-vonu~ -'° more stable. ,~ttty The high mass negative ion PD-mass spectrum (data not presented) showed the same fragmentation patterns with free acids as the methyl derivatives of MLA. Due to an abundance of adduct anions of lipid A, the spectrum of the methylated MLA was more difficult to interpret. The negative ion PD-mass spectrum in the lower mass range is shown in Fig. 6b. The fatty acyl anions originating from the cleavage of ester bonds are characteristic features of the spectrum. Anion peaks at m/z 187 and 227 correspond to the hydroxycapric acid (R~ and Rs) and myristic acid (R2), respectively. Fully and partially methylated phosphate anions appear in the spectrum with m/z 125 and 111.

Fragmentation pattern of DLA DLA's were prepared from the LPS of R. sphaeroides and R. capsulata by mild acid hydrolysis, purified by column chromatography, and analyzed by PD-MS. The partial positive ion PD-mass spectrum of the R. sphaeroides tetramethyl DLA is shown in Fig. 8. A molecular ion MNa + was observed at m / z 1578. A major fragment ion peak appeared at m/z 1470 corresponding to the loss of a methylated phosphate group, accompanied by H-transfer to the sugar. Because we did not observe a fragment ion corresponding to a loss of a phosphate group in MLA (see Fig. 7), it is likely that this is a loss of a ulvcosidic nhosphate ercmn_ A fragment ion (rdr~, _ pc~ _ H20) + at m/z 1452 was also observed. The loss of the phosphate group leads to dehydration and opening of the reducing-end sugar ring [21]. A minor ion peak at m/z 1726 might represent adduct formation between the sodium dimethyl phosphate (derived from the tetramethyl DLA) and the tetramethyl DLA. An examination of the low mass reg7 m of the negative ion spect,'um (Fig. 6c) revealed the absence of ~, trimethy! r'.,,,-'r,,.,-,~r,,,,~,. . . . . . h,-,,~,-,k,,,,~ anion at m/z 219. This suggested that the two phosphates in DLA are at different positions, and is consistent with the C-I and C-4' locations. The methylated MLA and DLA obtained from the LPS of R. capsulata were compared by PD-MS. The location of the fatty acyl groups in the lipid A from this source is established [22]- A9 - C+2OC+,~at the 3 '-position (RI), 3-oxo-Ci4 at the 2'-and 2-positions (R 2 and R 4, respectively~, and OHC+~; at the 3-position (R3) (refer to Fig. 3). The PD-mass spectra of methylated MLA and DLA are shown in Fig. 9. Dimethyl MLA and tetramethyl DLA produced mole+ 'lar ions (MNa + ~at m/z 1440 and 1548, respectively (Fig. 9a and 9b). The mass di! :nces of 171 and 108 corresponded to losses of OHC~0 and the dimethyl phosphate group (CHs)zPOs, respectively, as shown in Fig. 9a and 9b. Two-bond cleavage of the reducing-end sugar was observed ~

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[8, 9] to yield a peak at m/z 927. This cleavage was also observed with dimethyl MLA (Fig. 7). The significant point to be made here is that the loss of the dimethyl phosphate is not seen in the dimethyl MLA where a single phosphate group is at the 4'-position in the distal sugar. PD-MS is able to show only the loss of the reducing-end phosphate from the molecular ion as with tetramethyl DLA (Fig. 9b). The nature of the peak at m/z 1358 has not been determined (Fig. 9a).

164

Effect of KDO on fragmentation o f lipid A Hexaacyl ReLPS was isolated from E. coil D31m4, purified, methylated with diazomethane to the hexamethyl derivative and analyzed by PD-MS [14]. Several molecular ions were observed: (M + CH 2 + Na) +, (M + 2CH 2 + Na) + and (M + 3CH 2 + Na) + at m/z 2360, 2373 and 2387, respectively. A major fragmentation ion resulted from the loss of dimethyl phosphate from the reduc':ng end. This was followed by a clear loss of O-linked hydroxymyristoyl group, presumably from the non-stable 3-position of the disaccharide [5, 7]. Although poorly resolved, it indicated that myristoyl and lauroyi groups were lost. The presence of the two KDO units did not affect these reactions. However, the removal of the myristoxymyristoyl group was not detected. Since the KDO units are in close proximity to the ester-linked position of the distal sugar, this might protect the fatty acyl group at that position from mass spectral fragmentation. Conclusions As noted, the PD-MS method appears to combine fragmentation information observed previously in FAB-MS and infrared LD-MS. The presence of the oxonium ion, observed in FAB- and PD-MS, is critical to assigning the fatty acyl groups belonging to the distal glucosamine unit. Identification of the oxonium ion is, of course, aided by the presence of the phosphate group at the 4"position, so that the distal and reducing moieties are clearly distinguished in mass. Recently, for example, the structure of the lipid A from the LPS of Actinobacillus acetomycetemcomitans was reported [23], using PD-MS analysis of both intact (free acid) and dephosphorylated samples, the latter produced by HF treatment similar to the method employed by Seydel et al. [24] for the structural analysis of lipid A using LD-MS. Both samples produced abundant fragment ions from losses of ester linked fatty acids. However, I" . . . . . . . *~'~ ~:°'~' a--'~ ---~ " ~ sugars - are cnemlcally ' . . equivalent . . . in me de oh oso,.,.,~-~ ,,¢ u,~ta, , u ,cuucmg-cnu phorylated lipid A, it was not possible to assign any glycosidic fragments to either sugar on the basis of its spectrum alone. Additionally, it should be noted that because the phosphate carries two methyl groups in the derivatized case, one can unambiguously assign the oxonium ion in the mass spectra, based upon the shift of 28 amu. At the same time, two-bond ring cleavages are also observed by PD-MS and appear to arise from the MNa + (rather than MLI ÷ ions in analogy to the MK + ions generated in KCI doped LD-MS [8, 9, 16]. This is consistent with recent FAB-MS/MS studies by Orlando et al. [25] which suggest that two-bond ring cleavages are dominant in the product ion spectra of MNa + ions from carbohydrates, while glycosidic bond cleavages predominate in the product ion mass spectra of the protonated molecular ion. In any case, cleavages of the reducing sugar ring provide important information for assigning the N- and O-linked fatty acyl groups, and completing the structural analysis. Thus, PD-MS provides a rather complete approach for determini.g the basic structures of lipid A.

165

Acknowledgements This work was supported by the Research Service of the Veterans Administration, National Science Foundation Grants CHE-84-10506, BBS-85-15390 and National Institutes of Health Grant GM-36054. Mass spectra were obtained at the Middle Atlantic Mass Spectrometry Laboratory, a National Science Foundation Shared Instrumentation Facility, The Johns Hopkins University School of Medicine, Baiti. more, MD 21205, U.S.A. References 1 Galanos, C., Li.ideritz, O., Rietschel, E. Tb. and Westphal, O. (1977) Newer aspects of the chemistry and biology of bacterial lipopolysaccharides, with special reference to their lipid A component. Int. Rev. Biochem. 14, 239-335. 2 Morrison, D.C. and Ryan, J. L. (1979) Bacterial endotoxins and host immune response. Ad~,. lmmunol. 2 8 , 2 9 3 - 450. 3 Rietschel, E.Th., Ga!anos, C., L/ideritz~ O. and Westphal, O. (1982) Chemical structure, physiological funcnon and biological activity of lipopolysaccharides and lipid A. In: I mmunopharmacology and the Regulation of Leukocyte Function (Webb, D.R., Ed.) Dekker, New York, pp. 183 - 229. 4 Qureshi, N. and Takayama, K. (1990) Structure and function of lipid A. In: The Bacteria. A Treatise on Structure and Function (Iglewski, B.H. and Clark, V.L., Eds.) Vol. XI, Academic Press, San Diego, pp. 3 i 9 - 338. 5 Qureshi, N., Takayama, K., Heller, D. and Fenselau, C. (1983) Position of ester groups in the lipid A backbone of lipopolysaccharides obtained from Sahnonella typhimurium. J. Biol. Chem. 258, 12947 - 12951. 6 Takayama, K., Qureshi, N. and Mascagni, P. (1983) Complete structure of lipid A obtained from the lipopolysaccharides of the heptose-less mutant of Sahnonella typh#nurium. J. Biol. Chem. 258, 12801 - 12803. 7 Qurcshi, N., Cotter, R.J. and Takayama, K. (1986) Application of fast atom bombardment mass spectrometry and nuclear magnetic resonance on the structural analysis of purified lipid A. J. Microbiol. Methods 5, 6 5 - 77. 8 Takayama, K., Qureshi, N., Hyver, K., Honovich, J., Cotter, R.J., Mascagni, P. and Schneider, H. (1986) Characterization of a structural series of lipid A obtained from the lipopolysaccharides of Neisseria gonorrhoeae. Combined laser desorption and fast atom bombardment mass spectral analysis of high performance liquid chromatography-purified dimethyl derivatives. J. Biol. Chem. 261, 10624 - 10631. 9 Cotter, R.J., Honovich, J., Qureshi, N. and Takayama, K. (1987) Structural determination of lipid A from gram negative bacteria using laser desorption mass spectrometry. Biomed. Environ. Mass Spectrom. 14, 591 - 598. 10 Raetz, C.R.H., Purcell, S., Meyer, M.V., Qureshi, N. and Takayama, K. (1985) Isolation and characterization of eight lipid A precursors from a 3-deoxy-o-manno-octylosonic acid-deficient mutant of Sahnonella typhimurium. J. Biol. Chem. 260, 16080- 16088. 11 Torgerson, E. F., Skowronski, R. P. and Macfarlane, R. D. (1974) New approach to the mass spectroscopy of non-volatile compounds. Biochem. Biophys. Res. Commun. 60, 616- 621. 12 Macfarlane, R.D. and Torgerson, D.F. (1976) Californium-252 plasma desorption mass spectroscopy. Science 191,920-925. 13 Cotter, R.J. (1988) Plasma desorption mass spectrometry: Coming of age (review). Anal. Chem. 60, 781A - 793A. 14 Qureshi, N., Takayama, K., Mascagni, P., Honovich, J., Wong, R. and Cotter, R. J. (1988)Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia co6. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry. J. Biol. Chem. 263, 11971 - 11976. 15 Takayama, K., Qureshi, N., Mascagni, P., Nashed, M.A., Anderson, L. and Raetz, C.R.H. (1983)

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Fat.ty acyl derwatives of g!ucosamine l-phosphate in Eschetichia coli and their relation to lipid A. Complete structure of a diacyi GlcN-I-P found in a phosphatidylglycerol-deficient mutant. J. Biol. Chem. 258, 7379- 7385. Qureshi, N., Honovich, J.P., Hara, H., Cotter, R.J. and Takayama, K. (1988) Location of fatty acids in lipid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023. J. Biol. Chem. 263, 5592- 5504. Qureshi, N., Takayma, K., Meyer, K.C., Kirkland, T.N., Bush, C.A., Chen, L., Wang, R. and Cotter, R.J. (1991) Chemical reduction of 3-oxo and unsaturated groups in fatty acids of diphosphoryl lipid A from the iipopolysaccharide of Rhodopseudomonas sphaeroides. Comparison of ~;~loo;,-~! ,~,-,~n,.,-,;,~¢ before and ~¢t. . . . n,,..,;~,,, j Biol. c.h..... 266, ' : ~ " ,-¢~o Westphal, O. and Jann, K. (1965) Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5, 8 3 - 91. Raetz, C. R. H. (1987) Structure and biosynthesis of lipid A in Escherichia coli. In: Escherichia coli and Sahnonella o,phimurium. Cellular and Molecular ~iology (Ingraham, J . k . , Low, K.B., Magasanik, B., Schaechter, M. and Umbarger, E.H., Eds.) Vol. 1, American Society for Microbiology, Washington, DC, pp. 4 9 8 - 503. lmoto, M., Kusumoto, S., Shiba, T., Rietschel, E.T., Galanos, C. and Liideritz, O. (1985) Chemical structure of Escherichia coli lipid A. Tetrahedron Lett. 26, 907 - 908. Spengler, B., Dolce, J.W. and Cotter, R.J. (1990) Infrared laser desorption mass spectrometry of oligosaccharides and glycoside: Fragmentation mechanisms and isomer analysis. Anal. Chem. 62, 1731 - 1737. Krauss, J. H., Seydel, U., Weckesser, J. and Mayer, H. (1989) Structural analysis of the nontoxic lipid A of Rhodobacter capsulatus 3764. Eur. J. Biochem. 180, 519 - 526. Masoud, H., Weintraub, S., Wang, R., Cotter, R.J. and Holt, S.C. (1991) Investigation on lipid A structure of Actinobacillus acetinomycetemcomitans strains Y5 and human clinical isolate PO 1021-7. Eur. J. Biochem. 200, 7 7 5 - 781. Seydel, U., Lindner, B., Wollenweber, H.-W. and Rietschel, E.T. (1984) Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone. Eur. J. Biochem. 145,505 - 509. Orlando, R., Bush, C.A. and Fenselau, C. (1990) Structural analysis of oligosaccharides by tandem mass spectrometry: collisional activation of sodium adduct ions. Biomed. Environ. Mass Spectrom. 19, 747 - 754. *.aL|

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