©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Detailed Structural Analysis of a Novel, Specific O-Linked Glycan from the Prokaryote Flavobacterium meningosepticum(*)

Bruce B. Reinhold (1), Charles R. Hauer (2), Thomas H. Plummer (2), Vernon N. Reinhold (1)(§)

From the (1) Department of Nutrition, Harvard University School of Public Health, Boston, Massachusetts 02115 and the (2) Division of Molecular Medicine, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the preceding paper, preliminary analysis revealed a new type of O-linked oligosaccharide of 1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum (Plummer, T. H., Jr., Tarentino, A. L., and Hauer, C. R.(1995) J. Biol. Chem. 270, 13192-13196). In this report we detail the linkage, sequence, and branching of this unusual heptasaccharide by electrospray (ES) ionization mass spectrometry (MS), and collision-induced dissociation (CID). The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and CID. The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose. Only pyranose ring forms were detected ((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)GlcU-4[(2-OMe)Rham1-2]Man).


INTRODUCTION

In the course of studies on the hydrolases produced by Flavobacterium meningosepticum, eight major proteins have been purified, two of which were cloned and expressed in Escherichia coli, (endo--N-acetylglucosaminidases F and F). When compared on SDS-polyacrylamide gel electrophoresis, the native enzymes were slightly larger than their cloned counterparts and mass spectrometry (MS)() confirmed the products to be modified with carbohydrate during secretion. In the preceding paper (1) , these O-linked carbohydrates on P40 and endo--N-acetylglucosaminidases F and F appeared to be identical, and preliminary characterization revealed an oligosaccharide of 1244 Da at each of the proposed glycosylation sites. Collision-induced dissociation (CID) indicated an identical product ion series and compositional analysis revealed hexoses, methylated hexoses, and uronic acid derivatives (1) . Because of its unusual sugar composition, which included three different uronic acid derivatives, initial attempts at structural analysis of the Flavobacterium oligosaccharide by high resolution H NMR spectroscopy were unsuccessful in spite of the relative homogeneity (>80%) of the oligosaccharide chain. The complex spectrum of this unusual oligosaccharide was difficult to interpret in the absence of a suitable reference oligosaccharide. In addition, the well known resistance of uronic acid-containing polymers to acid hydrolysis and the instability of the amine-containing uronic acid greatly complicated the stoichiometric analysis of this oligosaccharide. An alternative approach using electrospray-MS (ES-MS) and CID provided a detailed structure for this O-linked heptasaccharide, including sequence, linkage, and branching. The structural assignments were supported by a combination of isotopic labeling, methylation, and composition analysis and the preparation of several chemical analogs and derivatives.


MATERIALS AND METHODS

P40 Glycopeptides

F. meningosepticum P40 and its tryptic glycopeptide were isolated and purified as outlined in the previous paper (1) . Intact oligosaccharide was released with concomitant reduction in 1 M NaBH in 50 mM NaOH at 45 °C for 16 h (2) .

Carbohydrate Analysis

Methylglycoside-trimethylsilyl Ethers and Acetates

Monosaccharide compositions from the glycopeptide, carboxyl-reduced glycopeptide, and previously released glycan were determined by gas-liquid chromatography following methanolysis and trimethylsilylation (3) and acetylation. All analyses were spiked with an internal standard (C-16 hydrocarbon), and retention times compared with commercial or synthetically prepared standards.

Alditol Acetates

Carbohydrate composition was also determined as alditol acetates prepared from the glycopeptide, carboxyl-reduced glycopeptide, and released glycan by gas-liquid chromatography following hydrolysis with 2.5 M trifluoroacetic acid at 120 °C for 2 h. The sample was neutralized with 0.1 M NaOH and reduced by the direct addition of 5 mg of solid NaBD, where D is deuterium (which remained at room temperature for an additional 16 h). Excess reducing agent was destroyed by the addition of 5 µl of acetic acid and the solution dried in a vacuum centrifuge. Borate was removed as its ester by repeated addition and drying with methanol. The sample was vacuum-desiccated overnight, and the dried residue was acetylated by the consecutive addition of 100 µl of pyridine and acetic anhydride and heated for 2 h at 75 °C. Gas-chromatography was performed on a Hewlett-Packard 5980 (Avondale, PA) using a 30 min 0.25-mm DB-5 column (J & W Corp.).

Methylation

Vacuum-desiccated glycopeptide, carboxyl-reduced glycopeptide, and released glycan were dissolved in 100 µl of a NaOH/MeSO suspension, prepared by vortexing MeSO and powdered sodium hydroxide (4) . After 1 h at room temperature, 35 µl of methyl iodide was added and the suspension set for 1 h at room temperature with occasional vortexing (5) . The methylated product was extracted into chloroform and back-washed with water until neutral. Methylation may be repeated to ensure complete derivatization.

Linkage Analysis as Methylated Alditol Acetates

Linkage analyses were performed following the established procedures of Lindberg et al.(6, 7, 8) , as modified by Geyer et al.(9, 10) . 16 µg of the methylated glycan (see above) was hydrolyzed with 160 µl of 2.5 M trifluoroacetic acid at 120 °C for 2 h. The product was directly dried by vacuum centrifugation and the residue reduced with 2.5 mg of NaBH in 125 µl of 0.1 M NaOH. This was heated to 65 °C for 1.5 h. The reaction was terminated with the addition of acetic acid and dried by vacuum centrifugation. Borate salts were removed as methyl esters by multiple addition of methanol and drying. This residue was acetylated by addition of 100 µl of pyridine and acetic anhydride and heated for 2 h at 75 °C. The products were again dried by vacuum centrifugation and extracted into chloroform. The products were identified and linkages determined by GS-MS.

Uronic Acid Esterification and Reduction

Esterification of carboxylate groups in polysaccharides (11) and proteins (12) have been described previously as well as subsequent reduction to the corresponding alcohol (11) . These procedures have been adapted for this glycopeptide by using 20 µg of lyophilized P40 dissolved in 125 µl of 1.8 N HCl in MeOH and letting set at room temperature for 1.5 h and then drying. The esterified residue was dissolved in 75 µl of methanol and reduced by the addition of 3 mg of NaBH and heated at 45 °C for 1.5 h. Excess reagent was decomposed by the addition of 10% acetic acid. Borate was removed as its ester by repeated addition and drying with methanol.

Periodate Oxidation and Reduction

Periodate oxidation (5, 13) was performed using a 9 mM solution of NaIO buffered with 0.1 M sodium acetate at pH 5.5 in a dark cold room (4 °C) for 3 days. The reaction was quenched with 3 µl of ethylene glycol and set overnight under the same conditions. The product was neutralized with 0.1 M NaOH and reduced by the direct addition of 2 mg of solid NaBH (which remained at room temperature for an additional 16 h). Excess reducing agent was destroyed by the addition of 5 µl of acetic acid and dried. Borate was removed as its ester by repeated addition and drying with methanol. The sample was vacuum-desiccated overnight prior to methylation.

Electrospray Ionization Mass Spectrometry

The instrument used in this study was a Finnigan-MAT TSQ-700 (Finnigan-MAT Corp., San Jose, CA) equipped with an electrospray ion source. Methylated samples were dissolved in methanol:water solutions (6:4, v/v) containing 0.25 mM sodium hydroxide and analyzed by syringe pump flow injected at a rate of 0.75 µl/min directly into the electrospray chamber through a stainless steel hypodermic needle. The voltage difference between the needle tip and the source electrode was -3.5 kV. For CID studies, multiply charged precursor ions were selectively transmitted by the first mass analyzer and directed into the collision cell containing argon at roughly 2 millitorr with acceleration voltages of 30-40 V, hence kinetic energies of 60-80 eV.


RESULTS AND DISCUSSION

Composition and Linkage Analysis

Composition analysis of the glycopeptide and reductively eliminated glycan had shown the presence of glucose, mannose (mannitol in reductively eliminated glycan samples), methylmannose, methyldeoxyhexose, and glucuronic acid. Further indication of uronyl residues was supported by a 42-Da increment to the glycan following methyl esterification, suggesting three carboxyl groups (1) . These compositions were confirmed by methanolysis of the glycopeptide (3) , but the sum of the peak areas was well below the anticipated mass of the glycan. To check for incomplete methanolysis, the reaction mixture was analyzed by ES-MS following acetylation (data not shown). The spectrum exhibited masses for the expected neutral monosaccharides (A-E) but, in addition, showed the presence of uronyl-containing disaccharides (F-H), a trisaccharide (I) (Fig. S1) and several unassigned higher mass ions. Interestingly, a methyluronyl residue was detected only in the disaccharide F. Thus, the poor quantification observed following composition analysis may be explained by the presence of uronyl residues that are relatively acid-stable (14) . To a lesser degree, 2-acetamidohexoses share the same properties so quantification of these components was greatly compromised.


Figure S1:



To offset the influence of uronyl residues, the carboxyl groups in the glycopeptide were esterified and reduced to the corresponding alcohol. Carbohydrate composition and linkage analysis of this carboxyl-reduced product identified two new components, 2-deoxy-2-acetamidoglucose and 2-O-methylglucose, indicating all three uronyl precursors to be glucuronyl analogs (glucuronic, 2-deoxy-2-acetamidoglucuronic, and 2-O-methylglucuronic). From these results, the glycan composition analysis suggested three hexoses (mannose, glucose, and 2-O-methylglucose), one deoxyhexose, (2-O-methylrhamnose),() and the three glucuronyl analogs for a total of seven unique structures. A repeat of the composition analysis following reductive elimination from the peptide detected the unique presence of mannitol and an absence of mannose providing evidence for O-linkage to the peptide. Linkage analysis, aided by CD-methylation (where D is deuterium), indicated two termini (2-O-methylmannose, 2-O-methylrhamnose) and a 4-O-linked glucosyl residue. Interestingly, no dibranched structures were detected (see below).

ES-MS-CID-MS of the Glycopeptide

Tryptic digestion of Flavobacterium P40 followed by gel filtration provided a single early eluting peak when analyzed by phenol-HSO and absorbance at 230 nm. Amino acid analysis of this glycopeptide fraction yielded the sequence S-I-L-D-S*-T-K, and this material served as the major source for subsequent experiments (1) . Analysis of this fraction by ES-MS produced prominent ions at m/z 1003.9 and 669.7, which was consistent with a doubly and triply protonated glycopeptide of 2006.8 Da. The peptide sequence (762.8 Da) leaves 1244 Da as the residue weight for the oligosaccharide, concordant with earlier results (1) .

Collision of the doubly charged ion, m/z 1004.1, provided the spectrum in Fig. 1, and ion intervals correspond to the residues identified in the composition analysis (e.g.m/z 162, 176, 190, 217 Da, hexose, methyl hexose or hexuronic acid, methyl uronic, and N-acetyl hexuronic acid, respectively). From this spectrum three series of glycosidic cleavage ions can be observed that suggest glycan sequence. One series (Fig. S2), can be followed as oligosaccharide increments to the protonated peptide (m/z 763) and the other two as decrements from the focused precursor (Fig. S3). Fragments from the oligosaccharide nonreducing end appear incomplete, although ions at m/z 394 and 732 do indicate some reducing end sequence information. Internal double cleavage ions, m/z 218 (GlcU) and m/z 556 (GlcUNAc-GlcU-Glc) were also detected and observed in the CID spectrum of the glycan alditol (1) . Increasing the collision energy resulted in some peptide fragmentation, but their intensities at all voltages were considerably less than those attributed to glycosidic cleavage.


Figure 1: Collision spectrum (CID) obtained from F. meningosepticum P40 glycopeptide. Focused precursor ion, m/z 1004.1, provided all singly charged products with the exception of m/z 807.4, 835.7, 916.0, and 925.1, representing nonreducing end losses (Schemes 2, 3, and 4).




Figure S2: Scheme 2




Figure S3: Scheme 3



Increments to the peptide ion, m/z 763 (Fig. 1) can be initiated by 162 Da for the fragment m/z 925, supporting a hexose moiety proximal to the peptide (Fig. S2) and consistent with the mannitol detected in the composition analysis of the reductively eliminated glycan. The subsequent intervals for methylrhamnose, methyluronic, hexose, uronic, N-acetyl uronic, and a methyl hexose residue suggest a linear sequence. But, this simple interpretation was compromised by the isomers of methylhexose and uronic acid and the inability to recognize double cleavage fragments that identify polymer branching.

Nonreducing terminal losses from the focused precursor, m/z 1004, provided two ion series initiated by a differential loss of 160 Da (Fig. 2, m/z 916.0, 925.1). Considering only one glycan has been identified, this suggests branching with a terminating methylrhamnose and a methylhexose residue (or the isomeric uronic acid).


Figure 2: Collision spectrum (CID) of the trisaccharide (B, Table I) obtained following basic methylation representing the reducing terminus fragment, m/z 633.3 (Scheme 7).



Alternatively, the upper fragment series may be explained by a secondary loss of 160 Da from each of the ions in the lower series (double glycosidic cleavage). Since the intact glycopeptide, and all ions of the lower branch, exhibit this loss, the residue must reside on the hexose moiety linked to the protein and be a 6-deoxyhexose, the methylrhamnose identified in composition analysis. Moreover, the lower branch (Fig. S3) intervals coincide with increments to the peptide (Fig. S2), again suggesting branching to be proximal to the peptide. Thus, the composition analysis combined with ES-MS and CID indicate the O-linked glycan to be a heptomer with a single branched methylrhamnose at the reducing end (Fig. S4).


Figure S4: Scheme 4



Polymer stability to acid hydrolysis, the lack of selected methylated alditol acetate standards, and the absence of some sequence ions in the CID spectra have compromised the above data. To compensate for these structural shortcomings and corroborate the proposed structure, further studies were initiated with the released oligosaccharide. In unrelated work with methylated oligosaccharides, CID has provided complete sequence, branching, and linkage information, the latter by detection of unique cross-ring fragments of the reducing side residue (15, 16) . When contrasting glycoproteins, glycopeptides, and methylated glycans, the latter CID spectra have proven to be more comprehensive and complete (17, 18) . Unfortunately, monomers with substituent groups (i.e. carboxyl, acetamido, amino, acetyl) do not show linkage-specific ions, and samples so substituted must be chemically modified.

ES-MS-CID-MS of the Glycan

Basic Deglycosylation

For glycan characterization, two strategies have been utilized for peptide deglycosylation, the classical base elimination methods of Carlson (2) , using hydride reduction to trap the alditol product, and direct base methylation (4) , as developed for O-linked peptides (3) . The former method provided the glycan as the alditol which was methylated (m/z 1566), whereas the latter procedure released and methylated a glycose analog in one step (m/z 1550). For the Flavobacterium P40 sample, both procedures resulted in extensive glycan degradation with the product spectra dominated by a major trisaccharide and several additional ions of lower abundance. The extensive degradation may be attributed to uronyl base-labile C-5 protons which causes a tautomeric shift in the -carboxyl group and -elimination of any C-4 residues (Fig. S5) (19) .


Figure S5: Scheme 5



Since esterification and sequence data had indicated the glycan to possess three internal uronyl moieties, one would anticipate the generation of a new methylglycoside and unsaturated uronyl terminus from each base-labile residue following methylation (Fig. S5). These partially eliminated products were observed when profiled by ES-MS and a comparison of the glycose and alditol product spectra provided further identification of all fragments possessing a reducing end (differ by 16 Da).

As an example, the methylated alditol exhibited a major ion at m/z 649 with additional ions at m/z 1330 and 1071, all shifted up by 16 Da, whereas the fragments m/z 954, 532, and 273 were invariant (Fig. S6). Additional ions were observed that could be accounted for as internal cleavage products (m/z 459, GlcUNAc-Glc, and m/z 718, GlcUNAc-GlcU-Glc). Thus, basic treatment of either glycan (aldose or alditol) appeared to yield the degradation products expected of internally linked, 4-O-hexuronyl residues, with six principle fragments each, three nonreducing (invariant) termini and three reducing terminal residues (Fig. S6).


Figure S6: Scheme 6



To clarify the positions of natural O-methylation, the reducing end trisaccharide was studied in greater detail with four chemical analogs. Differential methylation of these products provided unique ways to label specific fragments for structural identification. In addition to the alditol, three other samples were prepared, CH- and CD-methylated glycose, and a carboxyl ester back-exchange of the CD-methylated glycan by during CH-remethylation. These four analogs produced the corresponding trisaccharides, identified as A (m/z 649), B (m/z 633); C (m/z 654), and D (m/z 651), respectively (). CD-methylation identifies the number of methyl groups, differentiates natural methylation, and CH-remethylation characterizes carboxyl ester fragments by a 3-Da decrement. The collision spectra obtained from samples B and D are presented in Fig. 2and 3, and the major fragments for all samples are tabulated in . From these resulting mass shifts, fragmentation patterns have been summarized in Schemes 7 and 8.

The glycosidic loss of the uronyl residue from each trisaccharide analog (m/z 449 (A), m/z 433 (B), m/z 448 (C and D), ) and the corresponding mass shifts attributed to cross-ring fragmentation (m/z 505, 489, 504, and 504) identify the position of glucuronyl O-methyl substitution as carbon 2. In a similar manner, the cross-ring fragments (m/z 561, 545, 563, and 560), eliminate position 4 O-methyl substitution on methyl rhamnose, but fail to differentiate carbon 2 from 3. This was determined by periodate oxidation (below).

These data were again compatible with a branched trisaccharide at the reducing terminus. Since hexose analysis had identified mannitol following reductive elimination, the doubly linked hexose in this trisaccharide is of the manno configuration.

Glycan Periodate Oxidation, Reduction, and Methylation (ORM)

To further strengthen the linkage and sequence information, and localize indigenous methylation on each nonreducing terminus, the released alditol and glycopeptide were periodate-oxidized and reduced. Methylation again induced 4-O-elimination from uronyl residues (Fig. S5) and eliminated the oxidized glycan from the glycopeptide as the methylated glycose. The ES-MS profiles provided information on linkage and natural methylation (). No mass shifts were found on internal residues, whereas fragments containing a nonreducing terminal methylmannose exhibited a 2-Da increase. This increment would be compatible with natural methylation at position 2 or 4, since a 6-O-methyl group would cause a 44-Da decrement, whereas a 3-O-methyl group would leave the mass invariant. Indigenous methylation at position 2 was consistent with composition and methylation analysis.

ORM chemistry also modified the trisaccharide terminus and all fragments possessing this branched residue (). The glycose product (G-ORM, m/z 651, 1105, 1364, and 1570, ) showed a 2-Da increase, whereas the alditol product (A-ORM, ) was shifted down by 42 Da (e.g.m/z 607, 1061, 1320, and 1542). This latter difference may be considered the sum of a 44-Da decrement and a 2-Da increment. Collision analysis of both products indicated no change in the glucuronyl moiety (4-O-linkage and 2-O-methyl ether blocking periodate oxidation), whereas the methylrhamnose was responsible for the 2-Da increment.

The 2-Da methylrhamnose increment fixes natural O-methylation to carbon 2, since CID of the trisaccharide (, C or D) had identified either carbon 2 or 3, and the latter product would be resistant to oxidation. Of considerable interest was the 44-Da decrease attributed to the mannitol analog following ORM. This mass loss is consistent only with a combined 2-,4-branching pattern, since all other combinations would result in multiple carbon-carbon cleavages yielding different mass shifts (Fig. S9).


Figure S9: Scheme 9



Monomer Ring Form

Furanose ring forms are uncommon in most oligosaccharides, but are important structural differences not frequently considered. In this bacterial glycan all internal residues are 4-linked, and only the termini may be considered possible furanose candidates. A terminal methylrhamnofuranose would be unmodified following ORD, whereas a methylmannfuranose would show a 44-Da decrement. Since each termini showed only a 2-Da increment following ORM, they both must be pyrans. The peptide-linked mannose residue has been considered to be 2,4-linked from ORM studies; however, a 3,5-linked furan structure would satisfy both the ORM and CID sequence results, and this possibility has not been excluded.

Uronyl Ester Reduction

Reduction of uronyl esters within the polymer avoids the problem of alkali instability and provides a hexose analog for characterization and an oligosaccharide product suitable for CID linkage analysis by cross-ring cleavage. Thus, the Flavobacterium P40 glycopeptide was esterified (14) , reduced to the primary alcohol, and released by the separate steps of reductive elimination and methylation. Each sample provided a major doubly charged parent ion when analyzed by ES-MS with an absence of base degradation. Collision analysis of this analog released by methylation, m/z 765.6, provided the spectrum in Fig. 4.


Figure 4: Collision spectrum (CID) of the F. meningosepticum P40 glycan which was first prepared with uronyl carboxyl groups reduced and released by basic methylation (m/z 1508.7). Details of fragmentation defined in Scheme 10.



As expected, one major glycosidic cleavage dominated the spectrum which occurred adjacent to the acetamidohexose residue leaving a nonreducing terminal disaccharide fragment, m/z 486.4, and its pentasaccharide counterpart, m/z 1045.3. Smaller fragments define the sequence from both termini and four cross-ring fragments identify 4-O-linkages for each of four internal residues, m/z 329.1, 574.9, 778.4, and 982.4 (Fig. S10). A similar fragment to identify the penultimate GlcU-Man linkage was not expected or detected for the terminally positioned residue.


Figure S10: Scheme 10



ES-MS and CID supported by isotopic labeling and methylation analysis provide a powerful approach to structures not previously encountered in biological systems. In the present study, a new class of oligosaccharide has been discovered on several proteins secreted by the Gram-negative bacterium, F. meningosepticum. This simple prokaryote has a complex glycosyltransferase system which assembles a well defined highly acidic heptasaccharide at specific Asp-Ser or Asp-Thr-Thr consensus sites in several different secreted proteins. In this regard the system is similar to, but possibly predates, the attachment of oligosaccharides to consensus sites such as Asp-X-Ser/Thr sites in eukaryotic proteins.

There have been few other well characterized secreted prokaryotic glycans. The cellulolytic bacteria Clostridium thermocellum and Bacteroides cellulosolvens produce an extracellular complex containing O-linked glycans (20) . These differ from the acidic heptasaccharide produced by F. meningosepticum in two ways: 1) they are attached to the protein in Thr/Pro-rich regions but not at precise consensus sites, and 2) they are composed of short di-, tri-, and tetrasaccharides containing more typical components, 3-O-methyl-N-acetylglucosamine and galactose, with linkage via galactose. Other glycoproteins containing mannosyl-linked oligosaccharides have been described in eukaryotes but are structurally altogether different from the Flavobacterium acidic heptasaccharide. Yeast glycoproteins contain O-linked glycans with chains ranging from Man1 to Man5 that are attached in Ser/Thr-rich regions, but again with no obvious consensus site (21) . Mannose has also been reported as the linking sugar in keratin sulfate polysaccharides in the chondroitin sulfate proteolytic glycan of brain (22) , as well as in glucuronyl (16)mannosyl-O-threonine linkages in cuticle collagen of the clam worm (23) .

In summary the acidic heptasaccharide produced by F. meningosepticum is unique among both prokaryotes and eukaryotes. The function of this unusual acid heptasaccharide is unknown and is under investigation.

  
Table: CID of trisaccharide analogs

A = CH-alditol; B = CH-glycose; C = CD-glycoside; D = carboxylester back exchange of C by CH-methylation.


  
Table: Direct methylation (M), alditol ORM, and products

Glc = glucose; Man = mannose; Rham = rhamnose; GlcU = glucuronic acid; GlcUNAc = 2-acetamido-2-deoxy glucuronic acid; m = 2-O-methylated residue, e.g. mMan = 2-O-methylmannose; G-M = glycose-methylated; G-ORM = glycose, periodate-oxidized, -reduced, and -methylated; A-ORM = alditol: oxidized, reduced, and methylated.



FOOTNOTES

*
This study was supported in part by National Science Foundation Grants MCB-9400633 (to V. N. R.), NIH GM30471 (to T. H. P.), and NIH GM45781 (to V. N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Current address: Boston University Medical Center, Dept. of Microbiology and Immunology/Mass Spectrometry Resource, 80 East Concord St., Boston, MA 02118-2394. Tel.: 617-638-6762; Fax: 617-638-6763.

The abbreviations used are: MS, mass spectrometry; ES, electrospray ionization; CID, collision-induced dissociation; ORM, oxidation, reduction, and methylation; Glc, glucose; Man, mannose; Rham, rhamnose; GlcU, glucuronic acid; GlcUNAc, 2-acetamido-2-deoxy glucuronic acid.

This 2-O-methyldeoxyhexose was identified as the 2-O-methylrhamnose isomer by Dr. Roberta K. Merkle, University of Georgia.


ACKNOWLEDGEMENTS

The countless and precise chemical modifications carried out in this study were performed by Ms. Shui-Yung Chan for which we are most grateful.


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