Analysis of the core oligosaccharide of Aeromo 1 U 1 S hydrophila ( Chemotype III ) lipopolysaccharide using fast atom bombardment , electrospray and low energy tandem mass spectrometry

Fast-atom bombardment mass spectrometry (FAB-MS) was employed for the structural analysis of the core oligosaccharide of Aeromonas hydrophila (Chemotype III) lipopolysaccharide. Positive ion FAB-MS of the underivatized core oligosaccharide gave the protonated molecular ion, confrrming the correct composition in terms of hexoses, heptoses and Kdo which was present as a bicyclic furanosidic lactone. Negative ion FAB-MS gave the deprotonated molecular ion and fragment ions which were derived from more than two cleavage events with charge retention at the reducing and non-reducing terminals. Positive ion F AB-MS of the permethylated core oligosaccharide afforded fragment ions consistent with the defined sequence and branching patterns of the sugar constituents. The electrospray mass spectrum (ESMS) in the positive ion mode of the underivatized core oligosaccharide afforded the protonated molecular ion in the singly and doubly charged forms. Low energy collision-activated dissociation tandem mass spectrometry (CAD MS/MS) analysis of the protonated molecular ion [M


Introduction
Aeromonas hydrophila is a Gram-negative bacterium usually associated with outbreaks of disease in freshwater fish, particularly salmonids.More recently, it has been implicated in serious cases of human infection, which occasionally have resulted in death [1,2].Studies conducted on the serological identity of members of the Aeromonas hydrophiZa group indicated an extraordinary degree of heterogeneity [3].
Interest in the structure and immunological properties of the cell-surface polysaccharide of the different chemotypes of this Gram-negative bacterium has increased due to the fact that little is known about the biochemical basis of the pathogenicity of these species [4][5][6].
In the initial elucidation of the core oligosaccharide structure of Aeromonas hydrophila (Chemotype III), we presented an incomplete structure.This was due to the fact that, both in our original studies [4] and in the original chemotaxonomic classification of this lipopolysaccharide (LPS) core [7], we could not determine the presence of the a-D-Glc pN-(1 ~7)-L-a-D-Hep p disaccharide containing the D-glucosamine residue with a C-2 free amino group.The glycosidic linkage of this disaccharide was extremely resistant to the hydrolysis conditions normally used during sugar analysis and, therefore, eluded detection by conventional methods.Also, we were unable to detect 3-deoXY-D-manno-2-octulosonic acid (Kdo) which was considered a ubiquitous constituent of the inner core region of the lipopolysaccharide of the majority of Gram-negative bacteria.However, at the time of our initial investigation it was commonly believed that Kdo was absent from the lipopolysaccharides of the Vibrionaceae family and its absence, as established by the thiobarbiturate-based colorimetric assay, was used as a taxonomical characteristic for classification [8].
Subsequently, we have demonstrated that Kdo is present in all of the LPS belonging to the family 9, a result in direct contrast to the generally accepted view of the absence of this compolUld.The precise molecular structure of the core oligosaccharide from the cell surface has been revisited using state-of-the-art mass spectrometric techniques.It is well documented that mass spectrometry using surface ionization modes such as F AB is a powerful technique for the structural investigation of complex carbohydrates including those that contain labile and polar groups [10].Electrospray ionization (ES) is well established as a robust LC-MS technique that allows rapid, accurate and sensitive analysis of a wide range of analytes from low molecular weight polar compounds (less than 200 Da) to biopolymers larger than 100 kDa [11].
We now report on the application of F AB-MS and ESMS in the analysis of the structure of the core oligosaccharide of Aeromonas hydrophila (Chemotype III).The results of positive ion F AB-MS and ESMS of derivatized permethylated and N-acetylated permethylated products can be used to confirm the results obtained by negative and positive F AB-MS and positive ESMS of the underivatized oligosaccharide.We also report on the ring opening of the reducing Kdo which was found to be present in a bicyclic furanosidic lactone form, during the permethylation of the core oligosaccharide.

Results and discussion
In the recent course of structural investigation of the precise molecular structure of the lipopolysaccharide of the Gram negative bacterium of Aeromonas hydrophila (Chemotype ill), we isolated the disaccharide 7-0-(2-amino-2-deoxy-a-D-glucopyranosyl)-L-glycerO-D-manno-heptose 1, following hydrolysis of the respective core oligosaccharides with 2M hydrochloric acid for Ih at 100°C.The same aminoglycosylheptose 1 had been previously identified in the core region of the LPS of Escherichia coli [12], Bordetella pertussis [13] and Aeromonas hydrophila (Chemotype I and II) [5,6,14].Deamination of the native core oligosaccharide [5,6], followed by methylation analysis, established that the monosubstituted aminoglycosylbeptose 1 was substituted by an a-D-glucopyranosyl residue through the C-4 position of its heptose portion.When the core oligosaccharide of Aeromonas hydrophila (Chemotype III) was methylated by the Hakomori method, followed by hydrolysis with 2M trifluoroacetic acid, reduction, acetylation and identification by gas-    1), respectively.The presence of the glycosylalditol derivative 2 in this analysis could be attributed to the fact that this core oligosaccharide contained the trisaccharide heptapyranose which escaped detection in the previous investigation.Identification of 3-deoXY-D-manno-2-octulosonic acid (Kdo) was achieved by GC-EI-MS and it was found that this core oligosaccharide contained only one residue ofKdo which connected the native oligosaccharide to the Lipid A [9].We have suggested that the reducing Kdo was prone to fonn a lactone [15], and it was established that the single Kdo residue of this family of bacteria, which glycosidically attaches the core oligosaccharide to the non-reducing glucosamine unit of Lipid A, was in the furanose fonn [16,17] and linked through C-6.
On the basis of these new fmdings, combined with 2D NMR studies (COSY, DEPT and HETCOR), this core oligosaccharide was found to be composed of residues of Dgalactose, D-glucose, L-glycerO-D-manno-heptose, 3-acetamido-3,6-dideoXY-L-glucose, 2amino-2-deoxy-D-glucose and 3-deoXY-D-manno-2-octulosonic acid, in the approximate molar ratio of 1 :2:3: 1 : 1 : 1.The revised chemical structure of the core oligosaccharide of Aeromonas hydrophila, (Chemotype III), is as follows: Note that both the revised structure proposed in this rationale and the one presented in our previous studies would afford the same Smith degraded product, namely: Using positive ion F AB-MS, the native core oligosaccharide was determined to be C61HI02N2048 with a molecular weight of 1631.Using two different matrix mixtures composed of glyceroVthioglycerol (1 :3) and dithioerythritoVdithiothreitol (1 :5) we noticed a cluster of ions of which the most abundant were the protonated molecular ion [M+H]+ at mlz 1632 .andits l3C-isotope peak at mlz 1633.The ion at 1632 is consistent with the revised core structure which corresponds to Hex3HexNQui3NAcHep3Kdo, in which the Kdo furanose exists as 1,7-lactone.The protonated molecular adducts [M+H + thioglycerol] + and [M+H + dithioerythritol]+ and their BC-isotope peaks were also observed at mlz 1740, 1741, 1786 and 1787 respectively, as shown in Figure 2. It should be noted that except for molecular mass detennination, no useful structural sequence infonnation could be obtained from the F AB-MS using these two different matrices.
In contrast, the negative ion F AB-MS of the native core oligosaccharide, using a mixture of glycerollthioglycerol (1 :3) as matrix, showed the deprotonated molecular ion [M-H]-and its BC-isotope ion at mlz 1630 and 1631 respectively.Other diagnostic fragment ions were also observed which pennitted the confmnation of structural sequences of this branched core oligosaccharide (Figure 3).
The fragmentation routes of this negative ion F AB-MS, shown in Figure 4, have been rationalized using the scheme for systematic nomenclature for carbohydrate fragmentation in FAB-MS proposed by Domon and Costello [18,19] to describe putative fragmentation processes.Briefly, fragments with charge retention on the non-reducing terminus are designated A, B and C, while the reducing terminal fragments are similarly designated X, Y and Z. Fragments A and X contain superscript numbers designating the two bonds involved in ring cleavage which would account for the observed masses of these fragment series.Subscript numbers identify the residue number, while accompanying a,f3, etc., indicate the branch involved where a applies to the branch of highest mass.We have observed a series of "Y_type" fragment ions in which charge retention occurred on the reducing termirial end.Thus, the fragment ion [M-H-162l at mlz 1468 was attributed to the loss of one glucose residue from the deprotonated molecular ion and was assigned as We could also observe the simple Y 2 fragment ion [Hep(146)Kdof(I,7-lactone)J at mlz 411.It should be noted that charge retention was not restricted to one end of the molecule as demonstrated by the presence of "A-ring type cleavage" products in which more than one bond, often accompanied by molecular rearrangements [18,19], appears to split the same molecule as exemplified by the major fragment ions at mlz 766 and 426 tentatively assigned, respectively, as: The data obtained from the various fragment ions obtained by negative ion F AB-MS corroborates the majority of sequences proposed for the revised structure and confmns, once more, the presence of the Kdo furanose residue reducing end group which is present as aI, 7 -lactone.
The positive ion F AB-MS of the pennethylated core oligosaccharide prepared by Hakomori methylation using m-nitrobenzyl alcohol as a matrix is shown in Figure 5.The protonated molecular ion [M+H]+ at nv'z 2066 [~Hl(>4N2048' M Wt. 2065 Da] shows an increase of 434 Da resulting from the incorporation of 31 methyl groups, which is consistent with the core oligosaccharide structure Hex3HexNQui3NAcHep3Kdo(lactone) in which all hydroxyl, amine and amide groups are fully methylated.A major diagnostic fragment-ion resulting from multiple cleavages was observed at mlz 884.This fragment-ion was assigned to a matrix adduct of the protonated methylated trisaccharide Y3a+3<t+3P ion namely the [Hep(1~2)Hep(1~6)Kdof(lactone)+H+153]+ ion.We also noticed a series of diagnostic "B-fragment ions" resulting from charge retention at the non-reducing end at mlz 219, 230, 232, and 434 which were assigned respectively to the following pennethylated oxonium ions: [Glc]+, [Qui3NMeAc]+, [GlcNM~]+ and the disaccharide [Qui3NMeAc-(l~3)-GaI]+.Another important B diagnostic ion was observed at mlz 1153 and was assigned to the pentasaccharide ion having the following structure: Y3d+3P + B4 nv'z 1153 which resulted from the loss of the bicyclic Kdo furanosidic 1,7-lactone.The fragment ion at mlz 466 was assigned as the expected monohydroxylated disaccharide oxonium ion:  The fonnation of this fully methylated oxonium ion may be explained by an intennolecular reaction in the gaseous phase [20], between another methylated compolUld and the original monohydroxylated disaccharide oxonium ion at mlz 466.The tentative structw"es of these fragment ions are illustrated in Figure 6.
It is imperative to mention that in the F AB-MS of the pennethylated core oligosaccharide we did not observe the characteristic fragment ions resulting from the loss of "46" and "116" Da, a diagnostic pattern that has been observed only for core oligosaccharides that contain "normal" reducing pyranose Kdo residue [21].
We have also observed a cluster of higher masses resulting from increments of 14 Da and corresponding to a series of protonated molecular ions.Those were tentatively assigned as resulting from the lactone ring opening of the bicyclic furanosidic Kdo 1,7lactone.It has already been suggested by Dell and coworkers [21] that C-methylation of Kdo residue was possible and we propose that the bicyclic furanosidic Kdo 1,7-lactone may lUldergo a similar process.This may occur either as a result of ovennethylation or, possibly, by an intrinsically proper reaction resulting from the 1,7-lactone opening by the dimsyl anions as shown in Figure 6.The latter would result in the protonated molecular ions [M+H+Me]+ (C93H167N2048), [M+H+2Me]+ (C94HI6~2048)' [M+H+3Me]+ (~5H17IN2048) and [M+H+4Me]+ (C~173N2048) at mlz 2080,2094, 2108 and 2122 respectively, resulting from the incorporation of 1 to 4 extra methyl groups.These protonated molecular ions were tentatively rationalized as resulting fITst from opening of the lactone by the dimsyl anion to fonn consecutively the methyl ester followed by elimination of H-8 to afford the [M+H+Me]+ ion.This process is followed by C-methylation of C-8 to fonn the [M+H+2Me]+ ion, then C-methylation ofC-3 to form the [M+H+3Me]+ ion and fmally by another C-methylation of C-3 to form the [M+H+4Me]+ ion (Figure 6).
Electrospray MS in the positive ion mode afforded the spectrum shown in Figure 7.We could observe the singly charged protonated molecule [M+H]+ at mlz 1632 and the doubly charged diprotonated molecule [M+2H]+2 at mlz 817 reconfmning the molecular weight of this core oligosaccharide.Two singly charged fragment ions were also observed at mlz 1470 and 1283 which were assigned as [M+H-Glc]+ and [M+H-Glc-Qui3NAc]+.It should be noted that the ESMS was, indeed, cleaner than the F AB-MS and contained less fragments, no major background ambiguities and no matrix ions.Low energy tandem mass spectrometric analysis was conducted to rationalize the fragmentation pattern obtained in the conventional ESMS.Product ion spectra (also called daughter ion scan) arising from fragmentation in the RF-only hexapole collision cell of the quadrupole-hexapole-quadrupole instrument [22,23] was obtained.The [M+2H]+2 ion at mlz 817 was selected for the recording of the unimolecular mass-analyzed ion kinetic energy (MIKE) and collision activated dissociation MSIMS analyses.The CAD MSIMS of the [M+ 2H]+2 ion of the core oligosaccharide is presented in Figure 8 and suggests the formation of two major Y-product ions at mlz 1444 and 1283 assigned as [M+H-Qui3NAc]+ and [M+H-Qui3NAc-Hex]+, respectively.These two Y-product ions were the result of fragmentation with charge retention at the reducing end.Please note that in the CAD MSIMS of the [M+ 2H]2+ precursor ion, we observed the formation of the exclusive diagnostic ion [M+H-Qui3NAc]+ at mlz 1444 which was absent in the conventional ESMS.This low energy CAD MSIMS analysis provided additional confrrmation of the structure of this core oligosaccharide.

Experimental
Core oligosaccharide was prepared from the LPS of the parent bacteria, and gasliquid chromatography of the partially methylated alditol acetates and the peracetylated methyl glycosides was performed, as previously described [4].
F AB analyses (positive and negative ion mode) were performed on a VG ZAB2-SEQ mass spectrometer (VG Analytical Ltd, Manchester, U.K) with a BeqQ design, mass range 15 kDa, ion energy 8 kV, fitted with a cesium ion gun delivering approximately 2 JlA. of cesium ion current with approximately 35 kV energy.The matrix was either a mixture of glycerol / thioglycerol (1 :3) or a mixture of dithioerythritol / dithiothreitol (1 :5).
The ESMS (positive ion mode) were measured with a VG-Quattro quadrupolehexapole-quadrupole mass spectrometer, equipped with an electrospray ionization source, capable of analyzing ions up to m1z 4000.A 486, 66Iv1Hz personal computer equipped with Fisons MASSL YNX Mass Spectrometer Data System Software, was used for data acquisition and processing.The temperature of the ES ionization source was maintained at 70°C.The voltage of the ES capillary was 3.4 KV and the IN lens was at 0.4 KV throughout the whole operation.ESMS were obtained by scanning in the Multichannel Analysis mode (MCA) with a scan dwell time of 1 secondl250 a.m.u.. Spectra are an average of 3-4 scans.The mass scale was calibrated using a polyethylene glycol mixture (PEG 300/600/1000).MSIMS experiments were conducted on the same instrument.Fragment ion spectra of mass-selected ions were induced by collision with argon in the second (RF only) hexapole.Argon collision gas was added in the enclosed chamber of the hexapole to give a pressure of 2x10-5 mBar for collisional activation of the sample ions.The resulting fragments were analyzed by the second quadrupole.Collision energies of approximately 50 eV were used in all MSIMS experiments.

Conclusion
F AB-MS, ESMS and ES CAD MSIMS have assisted in the complete structural characterization and confrrmation of the revised molecular sequence of the core oligosaccharide of Aeromonas hydrophila, (Chemotype III) and constitutes an unique approach to the elucidation of more sophisticated natural bacterial polysaccharide structures containing the C-6 substituted furanosidic bicyclic 1,7-lactone fonn ofKdo.Both FAB-MS and ESMS (in the positive ion modes) showed that after methylation, the reducing tenninal furanosidic Kdo unit which is present in a 1,7-lactone (or furanosidic bicyclic 1,7-lactonized Kdo) affords a series of major components for which we have proposed tentative structures.We have postulated that the fonnation of such structures is the result of the opening of the 1,7-lactone of the reducing tenninal bicyclic furanosidic Kdo followed by a series of Cmethylations.It is also apparent that the characteristic molecular ion fmgetprint afforded by the four major products allows facile identification of core oligosaccharides containing a C-6 substituted reducing furanosidic Kdo residue prone to fonning 1,7-lactone which will escape detection by conventional hydrolytic and colorimetric methods of analysis.

Figure 3 .
Figure 3. Negative ion F AB-MS of the native core oligosaccharide of Aeromonas hydrophila (Chemotype III).

Figure 4 .
Figure 4. Fragmentation routes of the core oligosaccharide of Aeromonas hydrophUa (Chemotype ill) obtained in the negative ion FAB-MS.

Figure 6 .Figure 7 .
Figure 6.Chemical structure of the fragment-ions observed in positive ion FAB-MS of the pennethylated core oligosaccharide of Aeromonas hydrophiZa (Chemotype m).