Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide

Jianjun Li2, Sebastian H.J. Bauer3, Martin Månsson3, E. Richard Moxon4, James C. Richards2 and Elke K.H. Schweda1,3

2Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6; 3Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, S-141 86 Huddinge, Sweden; 4Molecular Infectious Diseases Group, University of Oxford Department of Pediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford 0X3 9DS, UK

Received on May 23, 2001; Revised on July 16, 2001; accepted on July 23, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A survey of both typeable and nontypeable strains of Haemophilus influenzae indicated that they contain glycine (Gly) in their lipopolysaccharide (LPS). Significant amounts (30–250 pmol Gly/µg LPS) were determined by high-performance anion-exchange chromatography using pulsed amperometric detection after treatment of the LPS with mild alkali. Oligosaccharides obtained from LPS after mild acid hydrolysis and gel filtration chromatography were investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis (CE) ESI-MS. In all cases, molecular ions corresponding to the major glycoforms were identified and were accompanied by ions differing by 57 Da, thus indicating the presence of glycine. The position of glycine in these glycoforms was determined by CE-ESI-MS/MS analyses. It was found that, depending on strain, glycine can substitute each of the heptoses of the inner-core element, L-{alpha}-D-Hepp-(1->2)-[PEtn->6]-L-{alpha}-D-Hepp-(1->3)-L-{alpha}-D-Hepp-(1->5)-{alpha}-Kdo of H. influenzae LPS as well as Kdo. In some strains, mixtures of monosubstituted Gly-containing glycoforms having different substitution patterns were identified.

Key words: Haemophilus influenzae/lipopolysaccharide/inner-core/glycine/CE-ESI-MS/MS


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Diseases caused by Haemophilus influenzae remain a significant problem worldwide. Type b capsular strains are associated with invasive diseases, including meningitis and pneumonia, while acapsular or nontypeable strains of H. influenzae (NTHi) are primary pathogens in otitis media and respiratory tract infections. Lipopolysaccharide (LPS) is an essential and characteristic surface component of these pathogens and is implicated as a major virulence factor. LPS of H. influenzae can mimic host glycolipids and has a propensity for reversible switching of terminal epitopes (phase variation) of the oligosaccharide portion. The conserved part of LPS from H. influenzae consists of a triheptosyl inner-core moiety in which each of the heptose residues (designated Hep I, II, and III) can provide a point for elongation by oligosaccharide chains or for attachment of noncarbohydrate substituents (Månsson et al., 2001Go and references therein) (Scheme 1).



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Scheme 1. Conserved part of LPS from H. influenzae. R1, R2, R3 = H or sugar residues.

 
To date, Hep I has been found to be substituted by glucose (R1 = ß-D-Glcp) in all strains investigated. There is no chain extension from Hep II in type d–derived strain Rd, however, in three type b strains and NTHi strain 486, Hep II is substituted by a glucose residue (R2 = {alpha}-D-Glcp), which can provide a point for further chain extension. Hep III is substituted by galactose (R3 = ß-D-Galp-(1->2); type b) or oligosaccharides extending from glucose (R3 = ß-D-Glcp-(1->2); Rd; R3 = ß-D-Glcp-(1->3); NTHi strain 486). Prominent noncarbohydrate substituents are phosphate (P), pyrophosphoethanolamine (PPEtn), phosphoethanolamine (PEtn), phosphocholine (PCho), and acetate (Ac). L-alanine, L-serine, L-threonine, and L-lysine have been found in several LPSs of, inter alia, Proteus where these were linked to uronic acids via amide linkages (Knirel and Kochetkov, 1994Go). Glycine (Gly) was detected in a survey of LPSs from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter, and Shigella species (Gamian et al., 1996Go). However, no linkage information has been reported for those strains. In addition, Gly was found to substitute a galacturonic acid residue in the core oligosaccharide of Plesiomonas shigelloides (Niedziela et al., 2000Go).

Mass spectrometry (MS) has become increasingly important in the structural analysis of lipolysaccharides (Masoud et al., 1997Go; Gaucher et al., 2000Go). The coupling of separation methods such as high-performance liquid chromatography and capillary electrophoresis (CE) with MS provides a powerful tool for rapid identification of target analytes present at trace levels in biological matrices and structural characterization of complex biomolecules (Thibault et al., 1997Go; Li et al., 1998Go). Recently we presented for the first time evidence for the presence of Gly in the LPS of H. influenzae nontypeable strain 486 (Månsson et al., 2001Go). In this study, high-performance anion-exchange chromatography using high-performance pulsed amperometric detection (HPAEC-PAD) and electrospray ionization mass spectrometry (ESI-MS) are employed to demonstrate that Gly is a common substituent in the H. influenzae LPS. In addition, information on the location of Gly in the LPS is provided by tandem mass spectrometry (MS/MS) following online separation by CE.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Analysis of Gly in H. influenzae LPS by HPAEC-PAD
When intact LPS of H. influenzae strain Rd was investigated by ESI-MS the corresponding spectrum revealed ions corresponding to earlier identified major glycoforms (Risberg et al., 1999aGo). In addition, ions were observed which differed from these by 57 Da. These ions were not present in O-deacylated LPS (LPS-OH) but pointed to the possibility that the LPS was further substituted by a base labile substituent on which information was lost due to experimental procedures. In fact, the main LPS structures in H. influenzae strain Rd had been established by us using nuclear magnetic resonance and ESI-MS on LPS-OH and major core oligosaccharides obtained after mild acid hydrolysis of LPS (Risberg et al., 1999bGo). In subsequent studies we observed minor ions that differed by 57 Da from major ions in ESI-MS spectra of oligosaccharides obtained after mild acid hydrolysis of LPS of NTHi strain 486 (Månsson et al., 2001Go). The presence of a single ester-linked Gly substituent in the corresponding glycoforms is consistent with these findings. To prove this, LPS from 24 NTHi and 5 typeable strains from H. influenzae were incubated with 0.1 ml of a 0.1 M NaOH solution at 22°C for 30 min. The reaction mixture was then analyzed by HPAEC-PAD for Gly. The 24 NTHi strains were chosen to represent the genetic diversity of NTHi after an analysis of the population structure by ribotyping (Bolduc et al., 2000Go). They were clinical isolates from the middle ear of patients with otitis media. For all strains a signal coeluting with authentic standard was detected corresponding to 30–250 pmol Gly/µg LPS (see Figure 1). Gly was present in all LPS preparations investigated.



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Fig. 1. Determination of Gly levels in 29 different strains of H. influenzae by HPAEC-PAD after incubation of LPS with 0.1 M NaOH. Quantifications were performed by comparison of peak areas with a calibration curve, which was obtained by chromatography of commercially available Gly. Numbers 486 to 1159 refer to the NTHi strains used. RM118[Rd] to Eagan refer to type d and b strains.

 
Analysis of Gly by CE-ESI-MS and MS/MS
To locate the position of Gly in the LPS, we employed CE-ESI-MS and MS/MS to investigate strain Rd (Risberg et al., 1999bGo) and NTHi strain 486 (Månsson et al., 2001Go) for which LPS structures have already been characterized. We found that online CE-ESI-MS provided high sensitivity and excellent resolution for the identificaton of LPS glycoforms. Coupled with MS/MS of the oligosaccharides in the positive ion mode, abundant fragment ions were obtained from cleavage of the glycosidic bonds, which provided information on the location of the Gly substituents. We then investigated the LPS from a selection of eight NTHi strains (162, 1233, 176, 1003, 1268, 1019, 1292, and 1200) for their Gly substitution by CE-ESI-MS/MS.

Gly linked to Hep III in strain Rd and NTHi 486
HPAEC-PAD showed strain Rd to contain 48 pmol Gly/µg LPS (Figure 1). Partial acid hydrolysis of LPS from H. influenzae strain Rd with dilute acetic acid in the presence of borane-N-methylmorpholine complex afforded the insoluble lipid A and reduced core oligosaccharide fractions, which were separated by gel filtration chromatography as previously reported (Risberg et al., 1999bGo). ESI-MS in the negative mode of the main fraction revealed a major doubly charged ion at m/z 785.5 corresponding to the major Hex3 glycoform (Scheme 2) of this strain.



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Scheme 2. The major Hex3 glycoform of H. influenzae strain Rd.

 
In addition, a signal at m/z 813.6 (<10% in ion abundance) corresponded to this glycoform containing a Gly substituent. Treatment of the core oligosaccharide fraction with 1 M NH4OH at 20–22°C for 16 h resulted in loss of this signal in the ESI-MS spectrum, confirming Gly to be ester-linked to the oligosaccharide part of LPS. The location of this substituent in the oligosaccharide was determined by CE-ESI-MS/MS in the positive ion mode. The product ion spectrum obtained from the doubly charged ion at m/z 816 and the proposed fragmentation pattern is shown in Figure 2A. The singly charged fragment ions at m/z 1057 and 574 corresponded to compositions PChoHexHep2PEtnAnKdo-ol and GlyHex2Hep, respectively, which was proven by further fragmentation of these ions. The MS3 spectrum of m/z 1057 is shown in Figure 2B. MS3 data was obtained by increasing the cone voltage to generate the precursor ion m/z 1057. The ion at m/z 328 corresponds to PChoHex, to which consecutive additions corresponding to Hep (m/z 520), AnKdo-ol (m/z 742), and HepPEtn (m/z 1057) are observed. In the MS3 spectrum on m/z 574 (Figure 2C) a significant ion at m/z 250 corresponding to HepGly is observed to which additions corresponding to Hex (m/z 412) and HexHex (m/z 574) are evident. These observations led to the conclusion that Gly substitutes Hep III in strain Rd.





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Fig. 2. CE-ESI-MS/MS (positive mode) analysis of the oligosaccharide from RM118 containing Gly. (A) Product ion spectrum of [M+2H]2+ m/z 816; the ion at m/z 654 is doubly charged and corresponds to a composition PChoGlyHexHep3PEtnAnKdo-ol. (B) MS/MS spectrum of fragment ion m/z 1057 promoted using an orifice voltage of 180V. (C) MS/MS spectrum of fragment ion m/z 574 promoted using an orifice voltage of 180V. The pattern of minor ions between prominent fragment ions at m/z 412 and 250 are likely to be due to consecutive losses of H2O (18 Da) and CH2O (30 Da) from m/z 412.

 
Recently we reported on the acetylated structure (Scheme 3) of the LPS oligosaccharide from NTHi strain 486, which is partly substituted by Gly (Månsson et al., 2001Go).



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Scheme 3. The acetylated structure of the LPS oligosaccharide from NTHi strain 486.

 
In the ESI-MS spectrum (negative mode) of the reduced oligosaccharide fraction obtained after mild acid hydrolysis and gel filtration chromatography, we observed ions at m/z 887.2 (base peak) and m/z 915.4 (20% in ion abundance), which corresponded to the major glycoform (composition: PChoAcHex4Hep3PEtnAnKdo-ol) and its glycinylated counterpart (composition: PChoAcGlyHex4Hep3PEtnAnKdo-ol), respectively. To locate the Gly residue CE-ESI-MS/MS (positive mode), experiments were performed on the corresponding doubly charged molecular ions at m/z 890 and 918, respectively. The product ion spectrum of the doubly charged ion at m/z 918 is shown in Figure 3A. Doubly charged ions at m/z 837, 756, and 675 could be attributed to consecutive losses of three hexose residues from the molecular ion. The singly charged ion at m/z 1219 was also observed in the CE-ESI-MS/MS experiment on m/z 890 (data not shown), and its composition PChoHex2Hep2PEtnAnKdo-ol was confirmed by an MS3 experiment (Figure 3B) showing consecutive losses due to Hex (m/z 1057), AnKdo-ol (m/z 835), Hep (m/z 643), PEtn (m/z 520), and Hep (m/z 328). Thus the loss of HexHexHepAcGly to give m/z 1219 clearly indicated Gly to be located at Hep III. Further evidence for this was observed in the ions at m/z 235 and 292 corresponding to HepAc and HepAcGly, respectively. The ion at m/z 292 was not observed in the CE-ESI-MS/MS spectrum of m/z 890.




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Fig. 3. CE-ESI-MS/MS (positive mode) analysis of the oligosaccharide from NTHi strain 486 containing Gly. (A) Product ion spectrum of [M+2H]2+ m/z 918. The doubly charged ion at m/z 645 corresponds to a composition of PChoAcGlyHex2Hep3PEtn due to consecutive losses of two Hex and a AnKdo-ol from m/z 918. (B) MS/MS spectrum of fragment ion m/z 1219 promoted using an orifice voltage of 180V. The ion at m/z 997 corresponds to loss of AnKdo-ol from m/z 1219. Minor ions at m/z 600 and 582 are likely due to consecutive loss of ethanolamine (43 Da) and H2O (18 Da) from m/z 643.

 
Gly substituting Kdo and Hep II in NTHi 162
The main oligosaccharide fraction obtained after mild acid hydrolysis of NTHi strain 162 was investigated by ESI-MS in the negative mode. The resulting spectrum revealed a major doubly charged ion at m/z 704.4 (base peak) corresponding to a composition of PChoHex2Hep3PEtnAnKdo-ol. In addition an ion at m/z 733.0 (8% in ion abundance) was observed corresponding to its Gly analogue, PChoGlyHex2Hep3PEtnAnKdo-ol. CE-ESI-MS in the positive mode revealed the corresponding ions at m/z 706.5 and 735, respectively. No structural data has been reported for the LPS of NTHi strain 162. To obtain information on the basic structure of the oligosaccharide in this strain and the position of Gly, ions m/z 706.5 and 735 were further fragmented in MS/MS experiments. The doubly charged ion m/z 706.5 showed ions corresponding to consecutive losses (data not shown) of Hex (m/z 1249) and Hep (m/z 1057) as well as ions at m/z 316, 328, 520, and 742 due to compositions HepPEtn, PChoHex, PChoHexHep, and PChoHexHepAnKdo-ol, respectively, which indicated the basic structure of the Hex2 glycoform (Scheme 4) in this strain.



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Scheme 4. The basic structure of the Hex2 glycoform in NTHi strain 162.

 
The MS/MS spectrum on the ion at m/z 735 due to the Gly-containing species is shown in Figure 4A. The spectrum contained an intense ion at m/z 280 that corresponded to a composition GlyAnKdo-ol, clearly indicating the Gly moiety to be substituting Kdo in this strain. This was corroborated by the ion at m/z 1189, which is due to loss of GlyAnKdo-ol from the molecular ion. The spectrum also revealed an ion at m/z 374 that corresponded to a composition of GlyHepPEtn, thus indicating an isomeric form in which Gly substituted Hep II. The occurrence of two isomeric forms was confirmed by further fragmenting m/z 1115, which is due to loss of HexHep from [M+2H]2+m/z 735. The resulting spectrum is shown in Figure 4B, which indicates fragment ions due to losses of either GlyAnKdo-ol or GlyHepPEtn at m/z 835 and 742, respectively.




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Fig. 4. CE-ESI-MS/MS (positive mode) analysis of the oligosaccharide from NTHi strain 162 containing Gly. (A) Product ion spectrum of [M+2H]2+ m/z 735, the ion at m/z 374 (marked with an asterisk) corresponds to a composition of GlyHepPEtn (B) MS/MS spectrum of fragment ion m/z 1115 promoted using an orifice voltage of 180 V.

 
Gly substituting Hep I in NTHi 1233
The main oligosaccharide fraction obtained after mild acid hydrolysis of NTHi strain 1233 was investigated by CE-ESI-MS/MS in the positive mode. The resulting spectrum revealed, inter alia, a major doubly charged ion at m/z 869 (base peak) corresponding to a composition of PChoHex4Hep3PEtnAnKdo-ol. In addition, a significant ion at m/z 897 (40% in ion abundance) was observed corresponding to its Gly containing counterpart, PChoGlyHex4Hep3PEtnAnKdo-ol. As for NTHi strain 162, no structural data was available for the oligosaccharide part of the LPS. MS/MS of the doubly charged ion at m/z 869 (data not shown) revealed a major ion at m/z 328 (HexPCho) with consecutive additions of Hep (m/z 520) and AnKdo-ol (m/z 742) as well as ions at m/z 1574, 1412, 1250, and 1058 due to consecutive losses corresponding to a Hex3Hep unit from the molecular ion. This was consistent with the structure (Scheme 5) for this Hex4 glycoform.



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Scheme 5. The structure for the Hex4 glycoform in NTHi strain 1233.

 
The CE-ESI-MS/MS spectrum on m/z 897 (Figure 5A) revealed ions at m/z 1630, 1468, 1306, 1115, and 1057 due to consecutive losses from a Hex3Hep or Hex3HepGly unit. The occurrence of both m/z 1115 and 1057 indicated the presence of isomers in which Gly substituted two different positions in the oligosaccharide. One position was clearly Hep III, which was corroborated by the ion at m/z 250 to which additions corresponding to two Hex residues could be made giving m/z 412 and 574, respectively. In addition, the MS3 experiment on m/z 574 (data not shown) was virtually identical with the spectrum shown in Figure 2C. The other substitution site of Gly was identified by further fragmenting the ion at m/z 1115 in an MS3 experiment. The MS3 spectrum on m/z 1115 (Figure 5B) showed losses due to AnKdo-ol (m/z 893) and HepPEtn (m/z 799). In addition the ion at m/z 799 showed a loss of AnKdo-ol (m/z 577). Thus ion m/z 577 could be attributed to a composition of PChoHexHepGly and implied the presence of an isomer in which Hep I is substituted by Gly.




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Fig. 5. CE-ESI-MS/MS (positive mode) analysis of the oligosaccharide from NTHi strain 1233 containing Gly. (A) Product ion of [M+2H]2+ m/z 897. (B) MS/MS spectrum of fragment ion m/z 1115 promoted using an orifice voltage of 180V.

 
The position of Gly in NTHi strains 176, 1003, 1268, 1019, 1292, and 1200
From the CE-ESI-MS/MS experiments performed on H. influenzae strains RM118, NTHi 486, NTHi 162, and NTHi 1233, the position of Gly was found to be on either Hep III, Hep II, Hep I, or Kdo. NTHi strains 1233 and 162 showed mixtures of substitution sites. The experiments led to the identification of specific fragment ions pointing to substitution sites: m/z 250 and 292 corresponded to HepGly and HepGlyAc, respectively, when Gly substituted Hep III; m/z 374 corresponded to GlyHepPEtn when Gly substituted Hep II. In addition, m/z 280 corresponded to GlyAnKdo-ol and was observed when Gly substituted Kdo. These "marker" ions were looked for in CE-ESI-MS/MS spectra of NTHi strains 176, 1003, 1268, 1019, 1292, and 1200 when molecular ions corresponding to glycoforms containing Gly were fragmented. Although specific marker ions were not evident when Gly was substituted on Hep I, the occurrence of this substitutent could be confirmed from examination of the fragment ions obtained following loss of Hep III and Hep II moieties. The results are summerized in Table I. It was found that Hep III is substituted by Gly in most of the strains investigated except strains 162 and 1200. Hep II is substituted in strain 162, and Hep I is substituted in strain 1233. Kdo is substituted in strains 162, 1003, 1268, 1292, and 1200. In addition, the appearance of several "marker" ions in the product ion spectra of some NTHi strains points to the occurrence of several LPS isomers in which Gly substitutes different positions in the inner core. Determination of the site of substitutions of Gly at Hep I, Hep II, HepIII, and Kdo is currently under investigation in our laboratory. It is particularly noteworthy that evidence for Gly substituting the outer core was not found in this investigation. The fact that Gly was found to either substitute Hep or Kdo and not Hex residues in the LPS oligosaccharides might suggest substitution at an exo-cyclic position. Preliminary evidence (data not shown) would suggest that this is the case at Hep III in the Hex4 LPS glycoform of NTHi 486.


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Table I. The positions of glycine in the inner core (Hep I–III, Kdo) of different H. influenzae strains
 

    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bacterial strains and LPS isolation
The NTHi strains were obtained from the Finnish Otitis Media Cohort Study and are isolates obtained from the middle ear. Bacteria were grown in Prof. E. R. Moxon’s laboratory (Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK) as described earlier (Månsson et al., 2001Go). The typeable H. influenzae strains RM118 (Rd), RM118-26, RM118-28, RM7004, and Eagan used in this study have been described earlier (Risberg et al., 1997Go, 1999a,b; Masoud et al., 1997Go; Weiser et al., 1997Go). In general, LPS was extracted from lyophilized bacteria by using phenol-chloroform-petroleum ether, as described earlier (Risberg et al., 1999bGo).

Chromatography
Gel filtration chromatography was performed using a Bio-Gel P-4 column (2.5 x 80 cm) with pyridinium acetate (0.1 M, pH 5.3) as eluent and a differential refractometer as detector. HPAEC-PAD was performed on a Dionex Series 4500i chromatography system (Dionex, Sunnyvale, CA) using a CarboPac PA1 column (4 x 250 mm) and pulsed amperometric detection. Gly was determined by HPAEC-PAD following treatment of LPS with 0.1 M NaOH (2.5 mg LPS/ml) at 22°C for 30 min. Twenty-five microliters of the reaction mixture were injected and eluted using a linear gradient of 0.1 M NaOH to 500 mM NaOAc in 0.1 M NaOH over 15 min and a flow rate of 1 ml/min. The retention time for Gly was 12.5 min. Quantifications were performed by comparison of peak areas with a calibration curve, which was obtained by chromatography of commercially available Gly (Sigma-Aldrich).

Preparation of oligosaccharides: mild acid hydrolysis of LPS
Reduced-core oligosaccharide material was obtained after mild hydrolysis of LPS (50 mg, 1% acetic acid, pH 3.1, 100°C, 2 h) in the presence of borane-N-methylmorpholine complex (7.0 mg) as the reducing agent. The insoluble lipid A was separated by centrifugation. The water-soluble part was purified by gel filtration chromatography, giving oligosaccharide-containing fractions which were investigated by ESI-MS.

MS
ESI-MS was performed with a VG Quattro Mass Spectrometer (Micromass, Manchester, UK) in the negative ion mode. Oligosaccharide samples were dissolved in water:acetonitrile (1:1) to a concentration of 1 mg/ml. Sample solutions were injected via a loop into a running solvent of water:acetonitrile (1:1) at a flow rate of 10 µl/min.

CE-ESI-MS and CE-ESI-MS/MS
A crystal Model 310 CE instrument (AYI Unicam, Boston, MA) was coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex, Concord, Canada) via a MicroIonspray interface. A sheath solution (isopropanol-methanol, 2:1) was delivered at a flow rate of 1 µl/min to a low dead volume tee (250 µm ID, Chromatographic Specialities, Brockville, Canada). All aqueous solutions were filtered through a 0.45-µm filter (Millipore, Bedford, MA) before use. An electrospray stainless steel needle (27 gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column. Separation were obtained on about 90-cm-length bare fused-silica capillary using 15 mM ammonium acetate/ammonium hydroxide in deionized waster, pH 9.0, containing 5% methanol. A voltage of 20 kV was typically applied at the injection end of the capillary. The outlet of the capillary was tapered to about 15 µm ID using a laser puller (Sutter Instruments, Novato, CA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full-mass scan mode. In the CE-ESI-MS, 30 nl of sample was typically injected by using 300 mbar for duration of 0.1 min. For CE-ESI-MS/MS experiments, about 60-nl sample was introduced using 300 mbar for 0.2 min. The MS/MS data were acquired in full scan mode using a dwell time of 2.0 ms per step of 1 m/z unit, which leads to the mass precision of ± 1Da. Fragment ions (formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell) were mass-analyzed by scanning the third quadrupole. In the triple quadrupole mass spectrometer, only MS2 or MS/MS is normally used for structural analysis. However, when the potential between the orifice plate and the skimmer is high enough, fragmentation can then take place. This method of collision-induced dissociation (CID) is referred to as front-end CID. In combination with the front-end CID, we are able to obtain MS/MS spectrum of a fragment ion or MS3, which was generated using an orifice voltage. In this study, the potential between the orifice plate and the skimmer was increased from 60 voltage to 180 voltage for MS3 experiments

The calculation of molecular mass values for proposed compositions were done using mass units as follows: Hex, 162.14; Hep, 192.17; AnKdo-ol, 222.20; PEtn, 123.05; PCho, 165.13; Ac, 42.04; and Gly, 57.05.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Derek W. Hood, Mary Deadman, and Shannon Walsh, Oxford, UK, are acknowledged for culture of nontypeable H. influenzae strains. S.H.J.B. gratefully acknowledges a Deutscher Akademischer Austauschdienst, Hochschulsonderprogramm III postdoctoral grant.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
AnKdo-ol, reduced anhydro Kdo; CE, capillary electrophoresis; CID, collision-induced dissociation; ESI-MS, electrospray ionization-mass spectrometry; Hep, L-glycero-D-manno-heptose; Hex, hexose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; LPS, lipopolysaccharide; MS/MS, tandem mass spectrometry; NTHi, nontypeable Haemophilus influenzae; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Gaucher S.P., Cancilla M.T., Phillips N.J., Gibson B.W, and Leary J.A. (2000) Mass spectral characterization of lipooligosaccharides from Haemophilus influenzae 2019. Biochemistry, 39, 12406–12414.[ISI][Medline]

Knirel, Y.A. and Kochetkov, N.K. (1994) The structure of lipopolysaccharides of gram-negative bacteria. III. The structure of O-Antigens: a review. Biochemistry (Moscow), 59, 1325–1383.

Li, J., Thibault, P., Martin, A., Richards, J.C., Wakarchuk , W.W., and van der Wilp, W. (1998) Development of an on-line preconcentration method for the analysis of pathogenic lipopolysaccharides using capillary electrophoresis-electrospray mass spectrometry. Application to small colony isolates. J. Chromatogr. A, 817, 325–336.[ISI]

Månsson, M., Bauer, S.H.J., Hood, D.W., Richards, J.C., Moxon, E.R., and Schweda, E.K.H. (2001) A new structural type for Haemophilus influenzae lipopolysaccharide: Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486. Eur. J. Biochem., 268, 2148–2159.[Abstract/Free Full Text]

Masoud, H., Moxon, E.R., Martin, A., Krajcarski, D., and Richards, J.C. (1997) Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b strain Eagan. Biochemistry, 36, 2091–2103.[ISI][Medline]

Niedziela, T., Czaja, J., Jachymek, W., Kenne, L., and Lugowski, C. (2000) A novel core oligosaccharide of Plesiomonas shigelloides devoid of phosphate residues. Poster abstract, 20th International Carbohydrate Symposium, Hamburg, Germany.

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