**3. Polysaccharides of** *Sinorhizobium meliloti*

Polysaccharides are commonly found at the surface of Gram negative bacteria (Figure 5). The aim of our work is to elucidate the structure of the polysaccharides from the surface of bacteria. Rhizobia are Gram negative bacteria living in soil and able to establish a symbiotic interaction with leguminous plants, known as nitrogen fixing symbiosis [90]. During this mutual interaction, bacteria bring combined nitrogen, in the form of ammonia directly transformed from atmospheric N2, to the plant. In turn the plant provides hydrocarbons and develops new organs on its roots which host the bacteria: the nodules [91,92]. During the early stages of establishing symbiosis, a molecular dialogue takes place. First, the partners in the soil are recognized. The plant exudes flavonoid compounds and the neighboring rhizobia respond by secreting Nod Factors (lipochitooligomers) [93]. Nod factors play a major role during the physical contact between the bacteria and the root hairs, and also trigger the organogenesis of the nodule [94]. Then the bacterial threads can invade the root to colonize the nodule. At this stage, the rhizobial surface polysaccharides are essential [95,96,97,98]. To enlarge our knowledge about their role, it is necessary to determine their

**Figure 5.** Scheme of the surface of Gram negative bacteria. The EPSs are not represented here; they are over the capsular polysaccharides.

structure. Their structural characterizations will be described below. They generally consist in analysis of composition by Gas Chromatography coupled with Mass Spectrometry (GC-MS), and sequence analysis made principally by Mass Spectrometry (MS). The choice of the MS coupling, mode or instrument depends on the nature of the polysaccharide (size and composition) as well as on the type of information we want to accede.

*Sinorhizobium meliloti* –the European model of rhizobia - has an external surface where 3 types of polysaccharides can be observed: exopolysaccharide (EPS), capsular polysaccharide (CPS) and lipopolysaccharide (LPS). Each class of PS have to be investigated alone, because their physico-chemical properties do not allow their simultaneous detection.

**Figure 6.** Isolation protocol for the different polysaccharides. In italic : caracterization methods.

## **3.1. Exopolysaccharides of** *Sinorhizobium meliloti*

#### *3.1.1. Isolation of the EPS*

324 The Complex World of Polysaccharides

over the capsular polysaccharides.

**3. Polysaccharides of** *Sinorhizobium meliloti*

separation.

Using a very simple 20 mM phosphate buffer (pH 8.5), the authors separated the different polysaccharides following the number of sialic acids, then MS and MS/MS spectra identified the composition of each polysaccharide thanks to (M+H)2+ or (M+H)3+ ions. An example of the mass electropherogram and the different mass spectra are presented in figure 4 which concerns the glycans from bovine fetuin. In this study the authors showed that this method can be used to identify polysaccharides from glycoprotein extracted from an SDS PAGE

Polysaccharides are commonly found at the surface of Gram negative bacteria (Figure 5). The aim of our work is to elucidate the structure of the polysaccharides from the surface of bacteria. Rhizobia are Gram negative bacteria living in soil and able to establish a symbiotic interaction with leguminous plants, known as nitrogen fixing symbiosis [90]. During this mutual interaction, bacteria bring combined nitrogen, in the form of ammonia directly transformed from atmospheric N2, to the plant. In turn the plant provides hydrocarbons and develops new organs on its roots which host the bacteria: the nodules [91,92]. During the early stages of establishing symbiosis, a molecular dialogue takes place. First, the partners in the soil are recognized. The plant exudes flavonoid compounds and the neighboring rhizobia respond by secreting Nod Factors (lipochitooligomers) [93]. Nod factors play a major role during the physical contact between the bacteria and the root hairs, and also trigger the organogenesis of the nodule [94]. Then the bacterial threads can invade the root to colonize the nodule. At this stage, the rhizobial surface polysaccharides are essential [95,96,97,98]. To enlarge our knowledge about their role, it is necessary to determine their

**Figure 5.** Scheme of the surface of Gram negative bacteria. The EPSs are not represented here; they are

The EPS are generally composed of many repetitions of 8 to 12-mers of hexose-subunits in a linear and/or branched form. This can be more or less substituted by O-acetyl, succinyl and pyruvyl groups. Their composition and structure is species specific and depends on the growth conditions [99]. The EPS are produced by the bacteria during the stationary phase of growth [99].

When the EPS of *S. meliloti* were studied, the cultures were stopped 5 hours after the stationary phase, by centrifugation. The supernatant, containing the EPS, was isolated and

lyophilized. When concentrated enough (increased viscosity but well dissolved), the EPS were precipitated, first with 3 volumes of ethanol. The pellet so formed was centrifuged and kept, the supernatant was concentrated. The pellet constitutes the high molecular weight (HMW) EPS fraction. The EPS left in the treated supernatant were then precipitated with 10 volumes of ethanol, the pellet was centrifuged and kept. This is the low molecular weight (LMW) EPS fraction. Each fraction was resuspended in water and subjected to proteinase K digestion (3.5 g/L at final concentration) for 4h at 36°C. The mixtures were then dialyzed against water. The pellets thus obtained were lyophilized, and once dried, used for structural analysis determination.

### *3.1.2. Characterization of the EPS*

To learn about the monosaccharide composition, the polysaccharides must be first hydrolyzed. This chemical reaction is carried out in acidic conditions. A solution of EPS in water was acidified by 98% TFA to a final concentration of 10%, and the mixture was kept at 100°C for 2h. At the end of the treatment, the leaving acid has to be evaporated. To help this elimination in a N2 stream, repetitive additions of isopropanol are necessary.

**Figure 7.** Chromatograms obtained by GC-MS for silylated standard saccharides and silylated hydrolyzed EPS LMW of *Sinorhizobium meliloti* 1021. EPS is made of Glucose and Galactose.

Coupled Mass Spectrometric Strategies for the Determination of Carbohydrates at Very Low Concentrations: The Case of Polysaccharides Involved in the Molecular Dialogue Between Plants and Rhizobia 327

326 The Complex World of Polysaccharides

structural analysis determination.

*3.1.2. Characterization of the EPS* 

lyophilized. When concentrated enough (increased viscosity but well dissolved), the EPS were precipitated, first with 3 volumes of ethanol. The pellet so formed was centrifuged and kept, the supernatant was concentrated. The pellet constitutes the high molecular weight (HMW) EPS fraction. The EPS left in the treated supernatant were then precipitated with 10 volumes of ethanol, the pellet was centrifuged and kept. This is the low molecular weight (LMW) EPS fraction. Each fraction was resuspended in water and subjected to proteinase K digestion (3.5 g/L at final concentration) for 4h at 36°C. The mixtures were then dialyzed against water. The pellets thus obtained were lyophilized, and once dried, used for

To learn about the monosaccharide composition, the polysaccharides must be first hydrolyzed. This chemical reaction is carried out in acidic conditions. A solution of EPS in water was acidified by 98% TFA to a final concentration of 10%, and the mixture was kept at 100°C for 2h. At the end of the treatment, the leaving acid has to be evaporated. To help this

elimination in a N2 stream, repetitive additions of isopropanol are necessary.

**Figure 7.** Chromatograms obtained by GC-MS for silylated standard saccharides and silylated hydrolyzed EPS LMW of *Sinorhizobium meliloti* 1021. EPS is made of Glucose and Galactose.

**Figure 8.** MS analyses of the LMW EPS of *Sinorhizobium meliloti* 1021A) ESI-MS analysis in negative mode B) ESI-MS/MS of ion *m/z* 1221.4 (the simplest form) amu. C) MS/MS of ion *m/z* 1321.4 amu.(the succinylated form), m/z 1383 corresponding to the succinylated and acetylated species.

After hydrolysis, the polysaccharides result in a mixture of monosaccharides and can be analyzed by GC-MS after derivatization. In the results presented here, we used the silylation of the sugar hydroxyl groups, which is a facile route. Actually, the protocol is simple, and a large databank is available for MS interpretations. In anhydrous pyridine, a mixture of HMDS and TMCS was added to the hydrolyzed EPS. This reaction was held for 30 min at 70°C [100]. After complete evaporation, the derivatized monosaccharides were injected directly as a hexane solution. The vector gas used was helium and the column was 95% dimethylsiloxane and 5% diphenylsiloxane. The temperature gradient was: 70°C hold for 3 min, increase at 5°C/min to 140°C and at 3°C/min to 240°C, then reach 300°C at 10°C/min, and hold at 300°C for 10 min.

This procedure applied to standard monosaccharides (glucose, galactose, mannose, glucuronic acid and galacturonic acid) allows interpretation of the chromatograms, determination of each sugar pattern (depending on alpha, beta, pyranoside, furanoside or lactone configuration) and the response factor for the different carbohydrates (Figure 7). The monosaccharide composition of the repeated units of LMW and HMW EPSs of *S. meliloti* was thus obtained.

The preparation of a simple solution of intact EPS in acidic methanol allows its ESI-MS analysis. This kind of analysis shows the purity of the EPS and the disparity of the sugars (Figure. 8A). Actually, the polysaccharide diversity is due to a variable degree of polymerization (DP) and to non-carbohydrate substituents. Here, the substituent is probably a succinyl group, adding 100 amu to ion *m/z* 1221.4 amu to yield the ion *m/z* 1321.4 amu. The ESI-MS/MS analysis of the ions found in the MS spectrum allows the sequence to be assessed by studying the fragmentation ions (Figure 8 B and C).

### **3.2. Lipopolysaccharides of** *Sinorhizobium meliloti*

LPS have three structural domains: the lipid A, the oligosaccharide core and the O-antigen polysaccharide. The LMW LPS, named rough LPS are composed of lipid A and core polysaccharides, whereas the HMW LPS (smooth LPS) are made of the 3 associated parts [101].

### *3.2.1. Isolation of the LPS*

As LPS are anchored in the outer membrane by lipid A, after growth of *S. meliloti* to the stationary phase, it was necessary to keep the pellet of centrifuged cultures (1L, grown until OD (600nm) 1.5). The pellet resuspended in about 40 mL of water was extracted by phenol at 60°C for 1h [102]. The extraction mixture was centrifuged and the upper phase (aqueous) contained the LPS, and all the other hydrosoluble molecules (DNAs, RNAs, proteins, carbohydrates). Enzymatic digestions were performed to eliminate DNAs, RNAs and proteins, remaining the carbohydrates. Affinity chromatography allowed the isolation of LPS. Then, gel filtration separated the LMW and HMW LPS (respectively rLPS and sLPS).

### *3.2.2. Characterization of the LPS*

328 The Complex World of Polysaccharides

and hold at 300°C for 10 min.

was thus obtained.

[101].

*3.2.1. Isolation of the LPS* 

After hydrolysis, the polysaccharides result in a mixture of monosaccharides and can be analyzed by GC-MS after derivatization. In the results presented here, we used the silylation of the sugar hydroxyl groups, which is a facile route. Actually, the protocol is simple, and a large databank is available for MS interpretations. In anhydrous pyridine, a mixture of HMDS and TMCS was added to the hydrolyzed EPS. This reaction was held for 30 min at 70°C [100]. After complete evaporation, the derivatized monosaccharides were injected directly as a hexane solution. The vector gas used was helium and the column was 95% dimethylsiloxane and 5% diphenylsiloxane. The temperature gradient was: 70°C hold for 3 min, increase at 5°C/min to 140°C and at 3°C/min to 240°C, then reach 300°C at 10°C/min,

This procedure applied to standard monosaccharides (glucose, galactose, mannose, glucuronic acid and galacturonic acid) allows interpretation of the chromatograms, determination of each sugar pattern (depending on alpha, beta, pyranoside, furanoside or lactone configuration) and the response factor for the different carbohydrates (Figure 7). The monosaccharide composition of the repeated units of LMW and HMW EPSs of *S. meliloti*

The preparation of a simple solution of intact EPS in acidic methanol allows its ESI-MS analysis. This kind of analysis shows the purity of the EPS and the disparity of the sugars (Figure. 8A). Actually, the polysaccharide diversity is due to a variable degree of polymerization (DP) and to non-carbohydrate substituents. Here, the substituent is probably a succinyl group, adding 100 amu to ion *m/z* 1221.4 amu to yield the ion *m/z* 1321.4 amu. The ESI-MS/MS analysis of the ions found in the MS spectrum allows the sequence to be

LPS have three structural domains: the lipid A, the oligosaccharide core and the O-antigen polysaccharide. The LMW LPS, named rough LPS are composed of lipid A and core polysaccharides, whereas the HMW LPS (smooth LPS) are made of the 3 associated parts

As LPS are anchored in the outer membrane by lipid A, after growth of *S. meliloti* to the stationary phase, it was necessary to keep the pellet of centrifuged cultures (1L, grown until OD (600nm) 1.5). The pellet resuspended in about 40 mL of water was extracted by phenol at 60°C for 1h [102]. The extraction mixture was centrifuged and the upper phase (aqueous) contained the LPS, and all the other hydrosoluble molecules (DNAs, RNAs, proteins, carbohydrates). Enzymatic digestions were performed to eliminate DNAs, RNAs and proteins, remaining the carbohydrates. Affinity chromatography allowed the isolation of LPS. Then, gel filtration separated the LMW and HMW LPS (respectively rLPS and sLPS).

assessed by studying the fragmentation ions (Figure 8 B and C).

**3.2. Lipopolysaccharides of** *Sinorhizobium meliloti*

In this work, it was the HMW LPS that were studied, so both types of structure had to be determined, the saccharide parts and the lipid A. Soft hydrolysis, in 1% acetic acid at 100°C for 1h, isolated lipid A, separating out of the aqueous solution. A liquid-liquid extraction, with CHCl3:CH3OH (3:1), separated the lipidic part from the sLPS.

Structural analysis of lipid A was performed by ESI-MS. The deconvoluted spectrum of doubly-charged ions revealed two series of monocharged ions, with a mass resolution that allowed the identification of the monoisotopic molecular masses (and not only the average molecular masses), i.e.: *m/z* 2050.41, 2038.39/2036.3, 2022.41, 2010.39 for one series and 1950.41, 1938.39/1936.3, 1922.37, 1910.37 for the second series with sufficient accuracy to allow elemental composition analysis (Figure 9, note that the spectrum presented is not deconvoluted). Accurate mass analysis combined with GC-MS data led to the proposal of a first general structure for lipid A : a di-phosphorylated penta-acylated diglucosamine comprising two 3OH-C14 fatty acids, one 3OH-C18, one 3OH-C19:1 and C27OH-C28 fatty acids for a compound giving a singly charged molecular ion of *m/z* 1950.4 amu (Figure 10)

**Figure 9.** A) Direct ESI-MS spectrum of LPS lipid anchor. Ions ranging from 954 to 976 are assumed to be molecular species and ions observed between *m/z* 1004 and 1026 are native methoxybutyrate derivatives. Insert B) high-resolution spectrum indicating the double charged state of lipidA.

The difference of 100 amu (50 amu in spectrum shown figure 9) can be attributed to the presence of a 3-methoxybutyrate in the first series. Actually, as the second lipid A distribution was hypothesized to be 3-O-methoxy-butyrate substitution of the 27OH-C28, exact mass measurements were performed. In the mean mass difference found between the respective members of the two distributions was 100.03 amu, corresponding with high significance to a C5O2H8. To confirm this hypothesis, MS/MS analyses of the double charged ion at *m/z* 1018.2 (Figure 11) have been performed. At low collision energy (15V) *m/z* 1018.2 generated three major fragments, respectively *m/z* 2004.4, 1992.4 and 1936.3 amu. *M/z* 2004 corresponds to the usual neutral loss of methanol (-32 amu), 1992 could be interpreted as a neutral loss of CO2 (44 amu) after rearrangement. Finally *m/z* 1936 corresponds to the ketene loss of the 3-methoxy butyrate. Increased collision energy (up to 40V) resulted in the production of the same fragments as observed directly from the parent ion that is not substituted by a 3-O-methoxy-butyrate (*m/z* 1935.4). The MS analyses of the complete LPS provided, by comparison to lipid A, the mass of the saccharide part.

**Figure 10.** General Lipid A structure of *Sinorhizobium meliloti* 1021's LPS. The dotted lines correspond to the substituent variation found in the sample.

Composition analysis of the saccharide part of the sLPS was performed as for the structural characterization of EPSs. After hydrolysis of the polysaccharide, the monosaccharides obtained were derivatized into alditol acetates, because the O-antigen usually exhibits great sugar diversity.

The process consisted of a reduction by sodium borohydride in ammonia for 1h at room temperature, followed by washing with acetic acid, and an acetic acid in methanol solution [103]. A supplementary wash was made in methanol only, before acetylation. After adding acetic anhydride and pyridine, the reaction stood for 1h at 70°C [104]. Liquid-liquid extraction in water against dichloromethane allowed the isolation of the so-formed alditol acetates in the organic phase. The dried mixture of derivatized monosaccharide was directly analyzed by GC-MS. The temperature gradient started at 110°C and increased at 3°C/min to reach 300°C. Of course, the same process was performed on standard monosaccharides to establish a short database, helpful for interpreting the chromatogram (Figure 12).

**Figure 11.** A) CID MS/MS high energy (Ecoll 40V) fragmentation of ion at *m/z* 1018 results principally in *m/z* 968 (loss of methoxybutyrate : -MeOBu) double charged species. B) deconvoluted spectrum indicating a characteristic methoxybutyrate fragmentation at lower collision energy (18V).

## **3.3. Capsular polysaccharides of** *Sinorhizobium meliloti*

In rhizobia, capsular polysaccharides are generally composed of a dimer repeating unit, composed of a hexose and a 1-carboxy-2-keto-3-deoxysugar, like Kdo (2-keto-3-deoxy octulosonic acid). Such a structure looks like the K-antigen found in *E. coli*, and was therefore named Kdo-rich capsular polysaccharide (KPS) for *Rhizobia* [105,106].

### *3.3.1. Isolation of the K type CPS (KPS)*

330 The Complex World of Polysaccharides

the substituent variation found in the sample.

sugar diversity.

respective members of the two distributions was 100.03 amu, corresponding with high significance to a C5O2H8. To confirm this hypothesis, MS/MS analyses of the double charged ion at *m/z* 1018.2 (Figure 11) have been performed. At low collision energy (15V) *m/z* 1018.2 generated three major fragments, respectively *m/z* 2004.4, 1992.4 and 1936.3 amu. *M/z* 2004 corresponds to the usual neutral loss of methanol (-32 amu), 1992 could be interpreted as a neutral loss of CO2 (44 amu) after rearrangement. Finally *m/z* 1936 corresponds to the ketene loss of the 3-methoxy butyrate. Increased collision energy (up to 40V) resulted in the production of the same fragments as observed directly from the parent ion that is not substituted by a 3-O-methoxy-butyrate (*m/z* 1935.4). The MS analyses of the complete LPS

**Figure 10.** General Lipid A structure of *Sinorhizobium meliloti* 1021's LPS. The dotted lines correspond to

Composition analysis of the saccharide part of the sLPS was performed as for the structural characterization of EPSs. After hydrolysis of the polysaccharide, the monosaccharides obtained were derivatized into alditol acetates, because the O-antigen usually exhibits great

The process consisted of a reduction by sodium borohydride in ammonia for 1h at room temperature, followed by washing with acetic acid, and an acetic acid in methanol solution [103]. A supplementary wash was made in methanol only, before acetylation. After adding acetic anhydride and pyridine, the reaction stood for 1h at 70°C [104]. Liquid-liquid extraction in water against dichloromethane allowed the isolation of the so-formed alditol acetates in the organic phase. The dried mixture of derivatized monosaccharide was directly analyzed by GC-MS. The temperature gradient started at 110°C and increased at 3°C/min to

provided, by comparison to lipid A, the mass of the saccharide part.

To isolate KPS from bacterial surface molecules, the same protocol as for the isolation of LPS was executed. Surprisingly, the *S.meliloti* 1021 KPSs were also retained by affinity chromatography. Gel filtration allowed enrichment in KPS, but their size is too close to the size of LPS, to be isolated from each other.

#### *3.3.2. Characterization of the EPS*

The analysis of carboxy-sugars such as Kdo is extremely difficult with GC-MS, due to their molecular weight and their instability. So, the composition of the KPS of *S. meliloti* was determined by GC-MS and mainly by HPAEC-PAD-MS. The latter technique requires an HPLC system, an anion exchange column and a desalter, which is required to couple the ion chromatograph with the MS (see part II.2.c). The device is detailed above, in the methodological part. Analysis of sugars with HPAEC needs no derivatization, better for the stability of Kdo. PAD detection is not sufficient to precisely determine the composition of the mixture analyzed, so the system is coupled to a mass spectrometer. The combination of HPAEC-PAD and MS is a challenge, because of the quantity of salt used in HPAEC-PAD, hence the presence of the desalter is essential. The challenge is rewarded by the information provided by this coupling for determining the saccharidic composition, with simple and non-destructive sample preparation.

**Figure 12.** Chromatograms obtained by GC-MS for alditol acetates of standard saccharides and alditol acetates of hydrolyzed LPS of *Sinorhizobium meliloti* 1021. LPS is made of Rhamnose, Glucose and Kdo.

For GC-MS analysis, as the acidic sugar content was high, derivatization with heptafluorobutyric anhydride (HFBA) was necessary [107]. The methanolyzed polysaccharides were dried and resuspended in anhydrous acetonitrile, a solution of HFBA was added. The reaction was heated to 60°C for 30 min. Evaporation of the leaving HFBA involved several co-evaporations with anhydrous acetonitrile. The final solution was made up in anhydrous acetonitrile and injected directly into the GC-MS. The temperature gradient was 70°C for 3 min, 5°C/min to 100°C, 3°C/min to 240°C and 5°C/min to reach 300°C and hold for 10 min. Figure 13 indicates that the KPS enriched fraction is exclusively composed of Kdo.

Coupled Mass Spectrometric Strategies for the Determination of Carbohydrates at Very Low Concentrations: The Case of Polysaccharides Involved in the Molecular Dialogue Between Plants and Rhizobia 333

332 The Complex World of Polysaccharides

non-destructive sample preparation.

HPLC system, an anion exchange column and a desalter, which is required to couple the ion chromatograph with the MS (see part II.2.c). The device is detailed above, in the methodological part. Analysis of sugars with HPAEC needs no derivatization, better for the stability of Kdo. PAD detection is not sufficient to precisely determine the composition of the mixture analyzed, so the system is coupled to a mass spectrometer. The combination of HPAEC-PAD and MS is a challenge, because of the quantity of salt used in HPAEC-PAD, hence the presence of the desalter is essential. The challenge is rewarded by the information provided by this coupling for determining the saccharidic composition, with simple and

**Figure 12.** Chromatograms obtained by GC-MS for alditol acetates of standard saccharides and alditol acetates of hydrolyzed LPS of *Sinorhizobium meliloti* 1021. LPS is made of Rhamnose, Glucose and Kdo.

For GC-MS analysis, as the acidic sugar content was high, derivatization with heptafluorobutyric anhydride (HFBA) was necessary [107]. The methanolyzed polysaccharides were dried and resuspended in anhydrous acetonitrile, a solution of HFBA was added. The reaction was heated to 60°C for 30 min. Evaporation of the leaving HFBA involved several co-evaporations with anhydrous acetonitrile. The final solution was made up in anhydrous acetonitrile and injected directly into the GC-MS. The temperature gradient was 70°C for 3 min, 5°C/min to 100°C, 3°C/min to 240°C and 5°C/min to reach 300°C and hold for 10 min. Figure 13 indicates that the KPS enriched fraction is exclusively composed of Kdo.

**Figure 13.** Chromatograms obtained by GC-MS for Kdo standard and methanolyzed KPS of *S. meliloti* 1021, both derivatized by HFBA. KPS is only composed of Kdo.

Unlike GC-MS analyses, in HPAEC-PAD-MS analyses, only soft hydrolysis of the polysaccharide mixture from the size exclusion chromatography (SEC) fractions containing KPS was implemented (1% acetic acid, 100°C, 1h). During the soft hydrolysis, a pellet appeared, indicating the presence of fatty acid on the KPS. MS analysis described bellow will detail this. The HPAEC-PAD-MS analysis revealed free Kdo and oligo-Kdo, characterized in MS by a mass difference of *m/z* 220 amu, when ion *m/z* 221 amu is extracted (Figure 14). The other compounds present in this sample are mostly composed of hexose dimers (*m/z* 324 amu), revealed when ion *m/z* 325 amu is extracted. So, the HPAEC-PAD chromatogram allows simultaneous analysis of other polysaccharides in this mixture: glycans, and substituted glycans [72].

A MS analysis confirmed that KPS is a homopolymer of Kdo. Actually, direct ESI-MS in negative mode (Figure 15 A) indicated the presence of many charged compounds. Zooming between *m/z* 1020 and *m/z* 1060, multi-charged ions (*e.g.*:1035.30 to 1036.05 are quadricharged ions and 1028.54 to 1029.04 are discharged ions) clearly appear (Figure 15 B). A deconvolution process recovers the native mass of the compounds (Figure 16 A). This reveals a mass difference of 220 amu between the members of the series, typically corresponding to Kdo. The MS/MS analysis of one of these polymers confirms that it is exclusively composed of Kdo, but also revealed the presence of an anchor explaining the discontinuity between Kdo15 and Kdo17 (Figure 16 B).

**Figure 14.** Chromatograms obtained by HPAEC-PAD-MS. A) Total ion current measured by MS. B) Chromatogram obtained when ion *m/z* 221 is extracted. C) Chromatogram obtained when ion *m/z* 325 is extracted. These chromatograms revealed that the KPS fraction is composed of polymers of Kdo and of glycans.

**Figure 15.** A) ESI-MS spectrum of KPS of *S. meliloti* 1021. B) Zoom on a group of ions revealing the multi-charged ions.

glycans.

multi-charged ions.

discontinuity between Kdo15 and Kdo17 (Figure 16 B).

corresponding to Kdo. The MS/MS analysis of one of these polymers confirms that it is exclusively composed of Kdo, but also revealed the presence of an anchor explaining the

**Figure 14.** Chromatograms obtained by HPAEC-PAD-MS. A) Total ion current measured by MS. B) Chromatogram obtained when ion *m/z* 221 is extracted. C) Chromatogram obtained when ion *m/z* 325 is extracted. These chromatograms revealed that the KPS fraction is composed of polymers of Kdo and of

**Figure 15.** A) ESI-MS spectrum of KPS of *S. meliloti* 1021. B) Zoom on a group of ions revealing the

**Figure 16.** A) deconvoluted spectrum of KPS of S.mmeiloti 1021. B) MS/MS analysis of one Kdopolymer, revealing an anchor on the KPS.

A MS/MS analysis of the smallest Kdo-oligomer revealed the mass of the anchor at *m/z* 622.4 amu. This ion has been studied separately by MS/MS, after mild hydrolysis on the KPS fraction (Figure 17). Ions m/z 530 corresponds to the loss of the glycerol and 548 to the loss of glycerol minus one water molecule. The interpretation of the spectra, in negative and in positive mode, determined that the anchor was lipidic and composed of a glycerol and phosphoglycerol unit, leading to the structure detailed in figure 18.

For structural characterization of polysaccharides, classical techniques are mostly used. Here, we reported results obtained with chromatography coupled to MS, MS alone and MS/MS. Depending on the characteristics, the type of polysaccharide analyzed, and which data are sought from those samples, it is necessary to adapt the coupled techniques. Advanced techniques, like NMR, are increasingly used for precise characterization. But NMR spectra of polysaccharides are very complex, therefore not easy to interpret. Actually, the heterogeneity in substitution and size is principally the origin of this complexity, especially in 2D NMR. Moreover, because of the interpretation difficulties, detailed mapping of NMR spectra of polysaccharides in the literature are seldom found. Lastly, as detailed in part I, note that polysaccharide solubility problems can lead to poorly resolved NMR spectra. While NMR can provide useful information about the polysaccharide, MS analysis is not obsolete, because the information provided with MS is unambiguous and confirms NMR spectrum interpretation. In many examples, MS analysis is necessary for an easier interpretation of the NMR spectra. Bacterial polysaccharides are highly complex molecules and many variations occur in one family. The role of polysaccharides from the rhizobia family during nitrogen fixing symbiosis has been demonstrated, as well as the activity of other bacterial polysaccharides during pathogen infection. However, little is known about their structure/activity relationships [108], which implies a long life for polysaccharide structural characterization.

**Figure 17.** Top, MS/MS analysis of the smallest compound containing Kdo in the KPS, revealing the anchor at *m/z* 622 amu (A). MS/MS Fragmentation of the lipidic anchor (ion *m/z* 622 amu) obtained after soft hydrolysis in negative (B) and positive (C) mode. The presence of glycerol and phosphoglycerol in this lipid anchor was thus demonstrated.

Coupled Mass Spectrometric Strategies for the Determination of Carbohydrates at Very Low Concentrations: The Case of Polysaccharides Involved in the Molecular Dialogue Between Plants and Rhizobia 337

**Figure 18.** General structure of KPS of *S. meliloti* 1021

## **Author details**

336 The Complex World of Polysaccharides

Moreover, because of the interpretation difficulties, detailed mapping of NMR spectra of polysaccharides in the literature are seldom found. Lastly, as detailed in part I, note that polysaccharide solubility problems can lead to poorly resolved NMR spectra. While NMR can provide useful information about the polysaccharide, MS analysis is not obsolete, because the information provided with MS is unambiguous and confirms NMR spectrum interpretation. In many examples, MS analysis is necessary for an easier interpretation of the NMR spectra. Bacterial polysaccharides are highly complex molecules and many variations occur in one family. The role of polysaccharides from the rhizobia family during nitrogen fixing symbiosis has been demonstrated, as well as the activity of other bacterial polysaccharides during pathogen infection. However, little is known about their structure/activity relationships [108],

**Figure 17.** Top, MS/MS analysis of the smallest compound containing Kdo in the KPS, revealing the anchor at *m/z* 622 amu (A). MS/MS Fragmentation of the lipidic anchor (ion *m/z* 622 amu) obtained after soft hydrolysis in negative (B) and positive (C) mode. The presence of glycerol and phosphoglycerol in

this lipid anchor was thus demonstrated.

which implies a long life for polysaccharide structural characterization.

V. Poinsot, M.A. Carpéné and F. Couderc *Laboratoire IMRCP, UMR 5623, Université Paul Sabatier, Toulouse, France* 

#### **4. References**



[23] Golovkina LS, Chizhov OS, Wul'fson NS Acetate of polyols. In"Mass spectrometric investigation of carbohydrate".vol 9. Bull. Acad Sci USSR, 1966.

338 The Complex World of Polysaccharides

[9] Bromund WH, Herbst RM (1945) The synthesis of oxazoline derivatives of monosaccharides and their relationship to the amino sugars. J. Org. Chem. 10: 267–276. [10] Kim JB, Carpita NC (1992) Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles, Plant Physiol. 98: 646-

[11] Maness NO, Ryan JD, Mort AJ (1990) Determination of the degree of methyl esterification of pectins in small samples by selective reduction of esterified

[12] Fontaine T, Fournet B, Karamanos Y (1994) A new procedure for the reduction of uronic

[13] Parkhomchuk AA, Kocharova NA, Białczak-Kokot M, Shashkov AS, Chizhov AO, Knirel YA, Rozalski A. (2010) Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens. O12.Carbohydr Res. 345: 1235-1239. [14] Wunschel DS, Colburn HA, Fox A, Fox KF, Harley WM, Wahl JH, Wahl KL (2008) Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis

[15] Lima LFO, Habu S, Gern JC, Nascimento BM, Parada JC, Noseda MD, Gonçalves A, Nisha VR , Pandey A , Soccol VT, Soccol CR (2008) Production and Characterization of the Exopolysaccharides Produced by Agaricus brasiliensis in Submerged Fermentation.

[16] Viccini G, Martinelli TR , Cognialli RCR, de Faria RO, Carbonero ER , Sassaki LK (2009) Mitchell DA Exopolysaccharide from surface-liquid culture of Clonostachys rosea

[17] Smiderle FR, Olsen LM, Carbonero ER, Marcon R, Baggio CH, Freitas CS, Santos ARS, Torri G, Gorin PAJ, Iacomini M (2008) A 3-O-methylated mannogalactan from Pleurotus pulmonarius: Structure and antinociceptive effect, Phytochemistry 69: 2731–

[18] Vesentini D, Steward D, Singh AP, Ball R, Daniel G, Franich R (2007) Chitosanmediated changes in cell wall composition, morphology and ultrastructure in two

[19] Marquesa G, Gutiérreza A, del Ríoa JC, Evtuguin DV (2010) Acetylated heteroxylan from Agave sisalana and its behavior in alkaline pulping and TCF/ECF bleaching. Carb.

[20] Ayestarán B, Guadalupe Z, León D (2004) Quantification of major grape polysaccharides (Tempranillo v.) released by maceration enzymes during the

[21] Quemener, Lahaye M, Metro F (1995) Assesment of methanolysis for the determination of composite sugars of gelling carrageenans and agarose by HPLC. Carbohydr. Res.

[22] Bleton J, Mejanelle P, Sansoulet J, Goursaud S, Tchapla (1996) Characterisation of neutral sugars and uronic acids after methanolysis and trimethylsilylation for

originates from autolysis of the biomass. Arch. Microbiol. 191:369–378

galacturonic acid to galactose. Anal. Biochem. 185 : 346-352.

spores, after culture. J. Microbiol. Methods. 74: 57-63.

Appl. Biochem. Biotechnol. 151:283–294

wood-inhabiting fungi. Mycol. Res. 111 :875-90.

fermentation process, Anal. Chim. Acta 513: 29–39.

recognition of plant gums, J Chromatogr. A 720: 27-49.

acid containing polysaccharides. J. Microbiol. Meth. 20: 149-157.

653.

2736

Polym. 81: 517–523.

266: 53-64.



340 The Complex World of Polysaccharides

1061-1064

4951-4959

analysis. Methods Mol. Biol. 534:53-64.

Mass Spectrom, 11: 1493-1504

Am. Chem. Soc., 113: 5964-5970

J. Am. Soc. Mass Spectrom, 13: 670-679

Commun mass Spectrom., 15: 2273-2283.

Spectrom., 9: 1125-1134

[41] Mechref Y, Kang P, Novotny MV. (2009) Solid-phase permethylation for glycomic

[42] Smiderle FR, Olsen LM, Carbonero ER, Marcon R, Baggio CH, Freitas CS, Santos AR, Torri G, Gorin PA, Iacomini M. A (2008) 3-O-methylated mannogalactan from Pleurotus pulmonarius: structure and antinociceptive effect. Phytochemistry, 69: 2731-2736. [43] Kim JS, Reuhs BL, Michon F, Kaiser RE, Arumugham RG. (2006) Addition of glycerol for improved methylation linkage analysis of polysaccharides. Carbohydr Res. 34:,

[44] Weiskopf A.S., Vouros P., Harvey P. (1997) Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry. Rapid Commun

[45] Visieux N, De Hoffmann E., Domon B. (1998) Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry. Anal. Chem., 70:

[46] Hofmeister G.E., Zhou Z., Leary J.A. (1991) Linkage position determination in lithiumcationized disaccharides: tandem mass spectrometry and semiempirical calculations J.

[47] König S., Leary J.A (1998) Evidence for linkage position determination in cobalt coordinated pentasaccharides using ion trap mass spectrometry J. Am. Soc. Mass

[48] Chai W., Lawson A.M., Piskarev V.J. (2002) The structural motif in chondroitin sulfate for adhesion of Plasmodium falciparum-infected erythrocytes comprises disaccharide units of 4-O-sulfated and non-sulfated N-acetylgalactosamine linked to glucuronic acid

[49] Domon B. and Costello C.E. (1988) A systematic nomenclature of carbohydrate fragmentation in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J., 5: 397-409 [50] Ngoka L.C., Gal J.F., Lebrilla C.B. (1994) Effects of cations and charge types on the

[51] Caroll J., Willard D., Lebrilla C. Energetics of cross-ring cleavages and their relevance to the linkage determination of oligosaccharides. Anal. Chim. Acta, 1995, 307: 431-447. [52] Dell A., Morris H.R., Egge H., von Nikolai H.Strecker G. (1983) Fast-atombombardment, negative-ion mass spectrometry of the mycobacterial O-methyl-D-

[54] Tseng K, Hedrick J.L., Lebrilla C.B. (1999) Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides, Anal Chem., 71: 3747-3754 [55] Zaia J. Mass spectrometric ionization of carbohydrates. In Encyclopedia of Mass Spectrometry, vol.6. M.G. Gross and r.M. Caprioli, eds. (Elsevier, New-York) [56] Deery M.J., Stimson E., Chappell C.G. (2001) Mass spectrometry and glycomics. Rapid

glucose polysaccharide and lipopolysaccharides. Carbohydr. Res., 115: 41-52 [53] McClellan J.M., Costello C.E., O'Connor P.B., Zaia J. (2002) Influence of charge state on product ion mass spectra and the determination of 4S/6S sulfation sequence of

metastable decay rates of oligosaccharides. Anal. Chem., 66: 692-698

chondroitin sulfate oligosaccharides. Anal Chem, 74: 3760-37771


anion-exchange chromatography with on-line ion-trap mass spectrometry. J. Chromatogr. B., 829: 136-143.


[85] Mechref Y (2011) Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry Electrophoresis, 32: 3467–3481

342 The Complex World of Polysaccharides

Chromatogr. B., 829: 136-143.

Chem., 62: 589A-597A.

251-255.

244: 283-290.

3820-3829.

Electrophoresis. 24: 3364-3370.

Anal Chem. 83: 5329-5336.

anion-exchange chromatography with on-line ion-trap mass spectrometry. J.

[71] Johnson D.C., Lacourse W.R. (1990) LC with pulsed ECD at gold and platinum Anal.

[72] Chataigné G, Couderc F, Poinsot V. (2008) Polysaccharides analysis of sinorhizobial capside by on-line anion exchange chromatography with pulsed amperometric

[73] Peter Katalinic J. (1994), Analysis of glycoconjugates by fast atom bombardment mass

[74] Treilhou M., Ferro M., Monteiro C., Poinsot V., Jabbouri S., Kanony C., Promé D., Promé JC. (2000) Differentiation of O-acetyl and O-carbamoyl esters of N-acetylglucosamine by decomposition of their oxonium ions: Application to the structure of

[75] Angel AS., Lindh F., Nilsson B. (2007) Determination of binding positions in oligosaccharides and glycosphingolipids by fast-atom-bombardment mass

[76] Berjeaud JM., Couderc F., Promé JC. (1993), Stereochemically controlled decomposition

[77] Grimshaw J (1997) Analysis of glycosaminoglycans and their oligosaccharide fragments

[78] Lagane B, Treilhou M, Couderc F (2000) Capillary Electrophoresis: Theory, Teaching Approach and Separation of oligosaccharides Using Indirect UV Detection BMBE, 28:

[79] El-Rassi Z, Postlewait J, Mechref Y, Ostrander GK. (1997) Capillary electrophoresis of carboxylated carbohydrates. III. Selective precolumn derivatization of glycosaminoglycan disaccharides with 7-aminonaphthalene-1,3-disulfonic acid fluorescing tag for ultrasensitive laser-induced fluorescence detection. Anal Biochem.

[80] Chen FT, Evangelista RA. (1995) Analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by capillary electrophoresis with

[81] Fraysse N, Verollet C, Couderc F, Poinsot V. (2003) Capillary electrophoresis as a simple and sensitive method to study polysaccharides of Sinorhizobium sp. NGR234.

[82] Szabo Z, Guttman A, Bones J, Karger BL. (2011) Rapid high-resolution characterization of functionally important monoclonal antibody N-glycans by capillary electrophoresis.

[83] Mittermayr S, Bones J, Doherty M, Guttman A, Rudd PM. (2011) Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation. J Proteome Res. 10:

[84] Szabo Z, Guttman A, Rejtar T, Karger BL. (2010) Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MS. Electrophoresis., 31: 1389-95.

detection and mass spectrometry coupling. J Chromatogr A. 1185:241-250.

spectrometry and related MS techniques, Mass Spectrom. Rev., 13: 77-98.

of silver-cationized methyl glycosides, Org. Mass Spectrom, 28, 455-458.

the non-reducing end of Nod factors. *JAS mass*, 11: 301-311.

by capillary electrophoresis Electrophoresis, 18, 2408-2414.

laser-induced fluorescence. Anal Biochem. 230: 273-280.

spectrometry. Carbohydr Res. 16:, 15-31.


