Confirmation of the Structure of Lipid A Derived from the

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Anal. Chem. 1997, 69, 2317-2322

Confirmation of the Structure of Lipid A Derived from the Lipopolysaccharide of Rhodobacter sphaeroides by a Combination of MALDI, LSIMS, and Tandem Mass Spectrometry Igor A. Kaltashov,† Vladimir Doroshenko,† Robert J. Cotter,*,† Kuni Takayama,‡ and Nilofer Qureshi‡

Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, Mycobacteriology Research Laboratory, W.S. Middleton Memorial Veterans Hospital, Madison, Wisconsin 53705, and Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

The chemical structure of nontoxic diphosphoryl lipid A from Rhodobacter sphaeroides was confirmed using a combination of LSIMS (on a two-sector mass spectrometer) and MALDI (on time-of-flight and ion trap mass spectrometers) in conjunction with tandem mass spectrometry in both positive and negative ion modes. Accurate molecular weight measurement accompanied by the analysis of fragment ion masses yielded the composition of fatty acyl groups. Tandem experiments (collisionally induced dissociation of both quasimolecular and oxonium ions) were also performed, revealing the precise location and nature of the fatty acyl groups on the disaccharide backbone. The lipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides has been shown to be a potent endotoxin inhibitor.1 The elucidation of the chemical structure of this lipid A was first attempted over a decade ago2 using a combination of TLC, electrophoresis, and GLC/MS. Although the composition of both the disaccharide backbone and the fatty acyl groups had been determined, the locations of the fatty acyl groups’ attachment sites had not been established. The structures of both monophosphoryl and diphosphoryl lipid A were later examined in more detail with the combination of laser desorption (LDMS), plasma desorption (PDMS), and fast atom bombardment (FABMS) mass spectrometry, aided by NMR analysis.3,4 Based on the results of these studies, a chemical structure of diphosphoryl lipid A from R. sphaeroides (Rs DPLA) was proposed in ref 4. The most interesting feature of this structure is the presence of 3-oxo and unsaturated fatty acyl groups (Figure 1). The validity of this structure has been recently disputed in the literature on the basis of minor differences in biological activities between the naturally occurring lipid A and the synthesized material.5,6 It was suggested that “the core substitution pattern is probably correct”, while the

Figure 1. Proposed chemical structure of tetramethyl diphosphoryl lipid A from R. sphaeroides.4 All stereochemical assignments are based on 1H NMR analysis.

assignment of the fatty acid side chains was questioned on the basis of TLC data.5 No mass spectral data were reported in this study.5,6 Recent advances in biochemical mass spectrometry, such as the introduction of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry,7 and further progress made in developing strategies for sequencing glycolipids by tandem mass spectrometry8-10 have enabled us to carry out detailed structural studies of complex lipopolysaccharides. In the present work, we have subjected tetramethyl Rs DPLA to scrupulous mass spectral analysis using soft ionization techniques such as LSIMS and

†

Johns Hopkins University School of Medicine. W.S. Middleton Memorial Veterans Hospital and University of Wisconsin. (1) Takayama, K.; Qureshi, N.; Beutler, B.; Kirkland, T. N. Infect. Immun. 1989, 57, 1336-1338. (2) Salimath, P. V.; Weckesser, J.; Strittmatter, W.; Mayer, H. Eur. J. Biochem. 1983, 136, 195-200. (3) Qureshi, N.; Honovich, J. P.; Hara, H.; Cotter, R. J.; Takayama, K. J. Biol. Chem. 1988, 263, 5502-5504. (4) Qureshi, N.; Takayama, K.; Meyer, K. C.; Kirkland, T. N.; Bush, C. A.; Chen, L.; Wang, R.; Cotter, R. J. J. Biol. Chem. 1991, 266, 6532-6538. ‡

S0003-2700(96)01294-2 CCC: $14.00

© 1997 American Chemical Society

(5) Christ, W. J.; McGuinness, P. D.; Asano, O.; Wang, Y.; Mullarkey, M. A.; Perez, M.; Hawkins, L. D.; Blythe, T. A.; Dubuc, G. R.; Robidoux, A. L. J. Am. Chem. Soc. 1994, 116, 3637-3638. (6) Christ, W. J.; Asano, O.; Robidoux, A. L.; Perez, M.; Wang, Y.; Dubuc, G.; Gavin, W. E.; Hawkins, L. D.; McGuinness, P. D.; Mullarkey, M. A.; Lewis, M. D.; Kishi, Y.; Kawata, T.; Bristol, J. R.; Rose, J. R.; Rossignol, D. P.; Kobayashi, S.; Hishimura, I.; Kimura, A.; Asakawa, N.; Katayama, K.; Yamatsu, I. Science 1995, 268, 80-83. (7) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301.

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Figure 2. Fractionation of tetramethyl DPLA from R. sphaeroides by reversed-phase HPLC. The preparation studied in the present work is marked with 1.

MALDI in conjunction with tandem mass spectrometry. All mass spectral data collected appear to be consistent with the originally proposed structure of Rs DPLA. EXPERIMENTAL SECTION Materials. R. sphaeroides ATCC 17023 was grown as described earlier.3 The procedures for extraction of the lipopolysaccharide content of the bacterial cell walls, its purification, and conversion to lipid A using mild hydrolysis have been described elsewhere.4 The highly purified pentaacyl Rs DPLA was converted to a free acid form, followed by methylation with diazomethane, yielding tetramethyl DPLA, and fractionated by reversed-phase HPLC.4 Fraction 1 (see Figure 2) was collected and subjected to a mass spectral analysis. Synthetic 2-hydroxydecanoic acid and 3-hydroxydecanoic acid were purchased from Matreya, Inc. (Pleasant Gap, PA) and used without further purification. Matrices for mass spectral analyses, triethanolamine (TEA) and 2,5-dihydroxybenzoic acid (gentisic acid), were purchased from Sigma Chemical Co. (St. Louis, MO); 3-nitrobenzyl alcohol (3-NBA) and niacin (nicotinic acid) were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI) (8) Costello, C. E.; Vath, J. E. In Methods in Enzymology; McCloskey, J. A., Ed.; Academic Press: San Diego, 1990; Vol. 193, pp 738-768. (9) Costello, C. E. In Biological Mass Spectrometry: Present and Future; Matsuo, T., Caprioli, R. M., Gross, M. L., Seyama, Y., Eds.; John Wiley and Sons Ltd.: New York, 1994; pp 437-462. (10) Chan, S.; Reinhold, V. N. Anal. Biochem. 1994, 218, 63-73.

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Mass Spectral Analysis. LSIMS spectra were acquired on a Concept IH (Kratos Analytical, Manchester, UK) two-sector (EB geometry) mass spectrometer at a resolution of 1000. Normally, a 1 µL aliquot of sample solution in methanol/chloroform (1:2) was mixed with the matrix (3-NBA) on the tip of the probe. Analyte ions were desorbed from the matrix by an 8 keV Cs+ primary ion beam. Mass spectra were acquired by scanning the magnet in the 100-2500 amu range at a scan rate of 10 s/decade. B/E linked scans were employed to acquire spectra of the product ions following high-energy collision-induced dissociation (CID, using Xe at 50% attenuation) of the precursor ions in the first fieldfree region of the mass spectrometer. Normally, 10-20 scans were signal-averaged for each spectrum. MALDI/time-of-flight (TOF) mass spectra were acquired on Kompact III (dual-stage reflectron) and Kompact IV (curved-field reflectron) time-of-flight mass spectrometers (Kratos Analytical, Manchester, UK). In most cases, gentisic acid was used as the matrix. Analyte ions were desorbed from the matrix by irradiation with a 337 nm pulsed nitrogen laser. Each spectrum was an average of 50 scans. MALDI/ion trap (IT) mass spectra were acquired on an extensively modified Finnigan MAT (San Jose, CA) quadrupole ion trap mass spectrometer described elsewhere.11 Analyte ions were desorbed from the matrix (nicotinic acid) with the fourth harmonic (226 nm) laser irradiation from a Nd:YAG laser (Quantel International, Santa Clara, CA). The low-energy CID spectra were acquired using broad-band excitation for precursor ion isolation and pulsed collision gas introduction to increase fragmentation efficiency.11 Ion trap data were processed on a PC using TOFWare (ILys Software, Pittsburgh, PA). RESULTS AND DISCUSSION Structural Information Based on MS1 Spectra. Molecular weight measurements of Rs DPLA were performed by LSIMS (on the two-sector instrument) and by MALDI (on the ion trap and the time-of-flight mass spectrometers) in the positive ion mode (Figure 3). The most abundant high-mass ion generated by LSIMS at m/z 1576.6 is in good agreement with the calculated monoisotopic mass (m/z 1576.9) for the species [M + Na]+ of Rs DPLA having the proposed structure. In addition, peaks at m/z 1590.6 and 1562.5 correspond to pentamethyl Rs DPLA and trimethyl Rs DPLA and are methylation byproducts. Other highmass ions represent loss of phosphate and/or fatty acyl groups (C10OH and C14:1) from the lipid A (Figure 3a). Desorption by MALDI apparently causes more facile fragmentation of the analyte; however, rather abundant quasimolecular ion peaks [M + Na]+ are present in both MALDI/TOF and MALDI/IT mass spectra (Figure 3b,c). The most abundant fragment ion peak in the MALDI/IT mass spectrum (m/z 872.5, B2) corresponds to an oxonium ion (calculated monoisotopic mass 872.6 amu) resulting from cleavage of the glycosidic bond. The oxonium ion peak is not present in the time-of-flight spectrum obtained in the reflectron mode, although it is rather abundant in the linear mode spectrum (data not shown). The oxonium fragment ion peak is also present in the LSIMS spectrum, along with a distal fragment ion ([0,4A2 + Na]+, following nomenclature by Costello8,9) at m/z 954.7 (calculated monoisotopic mass 954.6 amu). The two rather abundant ion peaks in the MALDI/IT mass spectrum (m/z 1014 and 1267) are not present in the LSIMS spectrum and are likely to result (11) Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1996, 68, 463-472.

Figure 3. LSIMS (a), MALDI/IT MS (b), and MALDI/TOF MS (c) positive ion spectra of Rs DPLA.

from intramolecular rearrangements of ions in the ion trap (vide infra) occurring during the significantly larger time scale of the ion trap analysis as compared with the other mass spectrometers (sector or time-of-flight). The two most abundant peaks in the negative ion LSIMS spectrum (shown in Figure 4) correspond to ions formed by deprotonation [M - H]- and demethylation [M - CH3]- of tetramethyl Rs DPLA. Other abundant high-mass ion peaks represent fragments due to loss of phosphate and/or fatty acyl groups. We note that the peaks at m/z 1198 and 1212 correspond to losses of two hydroxydecanoic fatty acyl groups (HOC10) from [M - H]- and [M - CH3]- ions of tetramethyl Rs DPLA, respectively. Other structurally informative fragment ions present in the spectrum represent the reducing end of the lipid A: Y1and Z1- (Y1- - H2O) at m/z 666 and 648, respectively. The distal part of the lipid A moiety is represented by a fragment ion at m/z 574 (B1- - HOC10 + H2O - HOPO3(CH3)2). The most abundant low-mass fragments in the negative ion LSIMS spectrum correspond to phosphate anions PO2(OCH3)2and PO3H(OCH3)- (m/z 111 and 125, respectively), as well as

carboxylate anions of hydroxydecanoic acid HOC10 (m/z 187) and tetradecenoic acid C14:1 (m/z 225). There is no evidence in the spectrum for the presence of anions of other fatty acids, hydroxy fatty acids, or a pyrophosphate anion. Structural Information Based on the Tandem Mass Spectra. The mass spectrum of the products of high-energy CID of the quasimolecular ion [M + Na]+ of Rs DPLA, generated by LSIMS, is shown in Figure 5. The most abundant fragment ions correspond to losses of phosphate and/or HOC10 and C14:1 fatty acyl groups. A peak at m/z 1324 corresponds to loss of two phosphate groups, confirming that the preparation used in this work is, indeed, diphosphoryl lipid A. The spectrum also contains a group of abundant fragment ion peaks corresponding to separation of the distal and reducing end subunits of the lipid A (the oxonium ion B1+ and the distal ion [0,4A2 + Na]+, as well as less abundant [B1+ - H + Na] and [C1 + Na]+ fragment ions). The two abundant lower-mass fragment ion peaks are likely to result from further decomposition of the oxonium fragment ion (consecutive losses of phosphate and hydroxycapric acid at m/z 558, and monounsaturated tetradecanoic acid at m/z 333). To prove this hypothesis (which would unambiguously assign HOC10 and C14:1 fatty acyl groups to the distal unit of the lipid A moiety), a low-energy CID mass spectrum of the oxonium ion was obtained on the ion trap instrument (Figure 6). Not surprisingly, the major fragment ions correspond to dissociation of a phosphate group and/or HOC10 and C14:1 fatty acyl groups from the precursor ion. This establishes immediately the fatty acid composition of the distal unit (O-linked HOC10 and C14:1, and N-linked HOC14). Further analysis of the spectrum (Figure 6) reveals a minor fragment ion peak, corresponding to a cross-ring cleavage at m/z 539 (2,4X+, assuming that HOC10 is attached to C-3′ and C14:1OC14 to C-2′, according to the structure previously proposed4). This same peak is also present in the LSIMS spectra, suggesting that the assignment of the fatty acyl groups in the distal unit is correct. This assignment also allows interpretation of the major fragment ion peak at m/z 1268 in the MALDI/IT mass spectrum (Figure 3b, vide supra) as [2,3X1 + Na - PO3C2H5]+. Further loss of a phosphate group or an HOC10 fatty acyl group results in appearance of two minor fragment ion peaks at m/z 1142 and 1081, respectively. The assignment of the fatty acyl groups attached to the reducing part of the lipid A is now rather straightforward. Since the lipid A has two O-linked HOC10 fatty acyl groups (the negative ion LSIMS data), and only one of them is attached to the distal sugar unit, the other must be present in the reducing part of the lipid A. This establishes the molecular weight of the N-linked fatty acyl group to be 451 amu, just 2 amu short of that for HOC14. Although the data presented here are not sufficient to establish the exact location of the unsaturation, the earlier conclusion that this fatty acid exists in a 3-oxo form4 seems to be well justified, since the N-linked fatty acids in lipid A molecules are known to be either (R)-3-hydroxy fatty acids (saturated) or 3-oxo fatty acids.12 It is possible now to interpret the fragment ion peak at m/z 1014 in the MALDI/IT mass spectrum (Figure 3b, vide supra) as [1,3A2 + Na - PO3C2H5]+. The most abundant fragment ion peaks in the high-energy CID spectrum of [M - CH3]- ions of the lipid A correspond to a loss of a phosphate group (HOPO3(CH3)2), as well as combined losses (12) Wollenweber, H.-W.; Rietschel, E. T. J. Microbiol. Methods 1990, 11, 195211.

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Figure 4. LSIMS negative ion spectrum of Rs DPLA. Inset: The low-mass region of the spectrum; peaks associated with the matrix are marked with *.

Figure 5. High-energy CID spectrum of [M + Na]+ (Rs DPLA).

of a phosphate group and either HOC10 or C14:1 fatty acyl groups (data not shown). The low-mass region of the spectrum also contains Y1- and Y1- - H2O fragment ion peaks representing the reducing end unit of the lipid A. Since O-linked fatty acids are known generally to exhibit more structural variation compared to N-linked fatty acids, negative ion 2320 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997

tandem mass spectrometry was employed to verify the nature of the O-linked fatty acyl groups. The low-mass region of the negative ion mass spectrum of the lipid A (insert on Figure 4) contained two peaks corresponding to fatty acids dissociated from the lipid A (this may be a result of fragmentation induced by the primary ion beam, as well as mild hydrolysis). The charge-remote

Figure 6. Low-energy CID spectrum of oxonium ion B1+ (Rs DPLA).

Figure 7. High-energy CID spectrum of a carboxylate anion at m/z 22+5 (C14:1- from Rs DPLA).

fragmentation of fatty acids is known to be greatly influenced by certain structural features,13,14 thus producing very informative fragmentation patterns. The high-energy CID spectrum of the carboxylate anion at m/z 255 (C14:1-) has been acquired on the sector instrument (Figure 7). The series of abundant fragment ions corresponding to losses of alkanes (-CnH2n+2) is sharply terminated at n ) 5, thus indicating that the double bond is located in the very middle of the chain (between C-7 and C-8). This allows a positive identification of the R2′ fatty acyl group as ∆7-C14:1, (13) Jensen, N. J.; Tomer, K. B.; Gross, M. L. Lipids 1986, 21, 580-588. (14) Gross, M. L. Int. J. Mass Spectrom. Ion Processes 1992, 118/119, 137-165.

confirming the proposed structure of the lipid A.4 Likewise, a high-energy CID spectrum of another carboxylate anion at m/z 187 (HOC10) has been acquired to establish the exact location of hydroxylation on the hydroxydecanoic fatty acyl chain of the lipid A moiety (Figure 8a). The presence of the hydroxy group on the fatty acid chain is known to influence the fragmentation pattern, although this influence is somewhat less straightforward compared to that of double bonds.15,16 To avoid possible confusion (15) Tomer, K. B.; Jensen, M. L.; Gross, M. L. Anal. Chem. 1986, 58, 24292433. (16) Kerwin, J. L.; Torvik, J. J. Anal. Biochem. 1996, 237, 56-64.

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the absence of these peaks in the spectrum in Figure 8c, established unequivocally that the hydroxy fatty acid derived from the lipid A moiety is, indeed, hydroxylated at C-3. The above analysis of the mass spectral data suggests that the preparation of Rs DPLA is a 1,4′-diphosphoryl pentaacyl lipid A. The fatty acyl groups attached to the distal sugar unit of the lipid A are 3-hydroxydecanoic acid (C-3′) and ∆7-[(tetradecenoyl) oxy]tetradecanoic acid (C-2′). The fatty acyl groups attached to the reducing sugar unit are 3-hydroxydecanoic acid (C-3) and oxotetradecanoic acid (C-2). This confirms that the covalent structure of Rs DPLA (Figure 1), proposed earlier,4 is correct. We have also noted that the preparation of Rs DPLA used in the present study appeared to be a rather stable compound, contrary to findings of Christ et al.5 The preparation was dissolved in methanol/chloroform mixture and kept at -5 °C, exhibiting only minimal sample degradation in the course of several months (mostly in the form of dissociation of anomeric phosphate and O-linked fatty acyl groups from the lipid A). CONCLUSIONS The covalent structure of the Rs DPLA preparation has been examined with a combination of LSIMS and MALDI MS. The accurate mass measurement of both quasimolecular [M + Na]+ ions and oxonium fragment ions allowed determination of the fatty acyl groups’ distribution between the distal and reducing glucosamine units of the lipid A. The nature of phosphate groups and the O-linked fatty acyl groups was established by analyzing the low-mass region of the negative ion spectrum, aided by the results of negative ion high-energy CID experiments. The positions of the fatty acyl groups on the disaccharide backbone were established on the basis of the results of tandem experiments (high-energy CID of the quasimolecular [M + Na]+ ions and lowenergy CID of the oxonium ions). The results of the present study suggest that the structure of Rs DPLA, reported earlier by Qureshi et al.,3,4 is correct. Tandem mass spectrometry, utilized on an ion trap (low-energy CID) and a sector instrument (high-energy CID), is shown to be an indispensable tool in structure elucidation of the lipid A moiety of lipopolysaccharides. Figure 8. High-energy CID spectra of carboxylate anions at m/z 187: HOC10- derived from Rs DPLA (a), 3-hydroxydecanoic acid (b), and 2-hydroxydecanoic acid (c).

in the assignment of the hydroxy group position, high-energy CID spectra of anions of synthetic 2-hydroxydecanoic acid and 3-hydroxydecanoic acid were also acquired (Figure 8b,c) and compared with the spectrum of carboxylate anion associated with the lipid A moiety. The appearance of two abundant fragment ion peaks at m/z 87 and 88, corresponding to a loss of heptane and heptyl from the precursor ion, in the spectra in Figure 8a,b, and

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ACKNOWLEDGMENT This work was supported in part by the grants from the National Institutes of Health, NIH-R01GM33967, NIH-R01RR08912, and GM50870. Received for review December 30, 1996. Accepted April 9, 1997.X AC9612943 X

Abstract published in Advance ACS Abstracts, June 1, 1997.