[methods in enzymology] glycobiology volume 480 || microbe-associated molecular patterns in innate...

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Microbe-Associated Molecular

Patterns in Innate Immunity:

Extraction and Chemical Analysis

of Gram-Negative Bacterial

Lipopolysaccharides

Cristina De Castro,* Michelangelo Parrilli,* Otto Holst,† and

Antonio Molinaro*

Contents

1. O

in

076

ersiersiionienc

verview

Enzymology, Volume 480 # 2010

-6879, DOI: 10.1016/S0076-6879(10)80005-9 All rig

ta di Napoli Federico II, Dipartimento di Chimica Organica e Biochimica,tario Monte Santangelo, Via Cynthia, Napoli, Italyof Structural Biochemistry, Research Center Borstel, Leibniz-Center for Mes, Borstel, Germany

Else

hts

C

edic

91

2. L
PS and LOS Extraction Procedures 93
2
.1. P CP extraction of LOS 94
2
.2. M ethods 94
2
.3. P henol/TEA/EDTA extraction 95
2
.4. M ethods 95
2
.5. H ot phenol/water extraction 95
3. P
urification of the Crude Extracts 96
3
.1. E nzymatic hydrolysis 96
3
.2. M ethod 97
4. S
DS-PAGE 98
4
.1. K ittelberg and Hilbink protocol 98
4
.2. M ethods 98
4
.3. T sai and Frasch (1982) protocol 99
4
.4. M ethod 100
4
.5. D etection of acidic polysaccharides 100
4
.6. M ethod 100
5. C
arbohydrate Analysis: Monosaccharide Determination, Absolute
Configuration, and Definition of Branching Points
101
5
.1. M onosaccharide determination: Acetylated methyl glycosides 101
5
.2. M ethod 102
vier Inc.

reserved.

omplesso

ine and

89

90 Cristina De Castro et al.

5
.3. M onosaccharide absolute configuration 103
5
.4. M ethods 104
5
.5. D etermination of the absolute configuration of carbohydrates
in a sample
105
5
.6. D etermination of monosaccharides branching points
(methylation analysis): protocol for neutral and uronic acid

containing polysaccharides
105
6. F
atty Acids Compositional Analysis (GC-MS) 109
6
.1. T otal fatty acid composition by methanolysis 109
6
.2. M ethods 112
6
.3. O -Linked fatty acid 112
6
.4. M ethods 112
6
.5. A bsolute configuration determination of hydroxyl fatty acids 113
6
.6. M ethods 113
Refe
rences 114
Abstract

Bacterial lipopolysaccharides (LPSs) are the major component of the outer

membrane of Gram-negative bacteria. They have a structural role since they

contribute to the cellular rigidity by increasing the strength of cell wall and

mediating contacts with the external environment that can induce structural

changes to allow life in different conditions. Furthermore, the low permeability

of the outer membrane acts as a barrier to protect bacteria from host-derived

antimicrobial compounds. They also have a very important role in the elicitation

of the animal and plant host innate immunity since they are microbe-associated

molecular patterns, namely, they are glycoconjugates produced only by Gram-

negative bacteria and are recognized as a molecular hallmark of invading

microbes. LPSs are amphiphilic macromolecules generally comprising three

defined regions distinguished by their genetics, structures, and function: the

lipid A, the core oligosaccharide and a polysaccharide portion, the O-chain. In

some Gram-negative bacteria, LPS can terminate with the core portion to

form rough-type LPS (R-LPS, LOS). In this chapter, we will describe the isolation

of both kinds of LPSs and their full chemical analysis, pivotal operations

in the complete description of the primary structure of such important

glycoconjugates.

Abbreviation list

Kdo
3-deoxy-D-manno-oct-2-ulosonic acid DMSO dimethylsulfoxide

Analysis of Bacterial Lipopolysaccharides 91

e.i.
electronic impact EDTA ethylendiamine tetracetate GC-MS gas chromatograph equipped with mass spectrometer Glc glucose LOS lipooligosaccharide LPS lipopolysaccharide ML McLafferty M molecular ion AAPM partially methylated and acetylated alditol PCP phenol/chloroform/light petroleum extraction PAGE polyacrylamide gel electrophoresis SDS sodium dodecyl sulfate TEA triethylamine
1. Overview

Gram-negative bacterial lipopolysaccharides (LPSs) are powerfulvirulence factors and among the most important elicitors of eukaryoticinnate immune response both in animals and plants (Alexander andRietschel, 2001; Raetz and Whitfield, 2002, Silipo et al., 2010). Theyare heat-stable complex amphiphilic macromolecules of the Gram-negativecell envelope, and indispensable for bacterial growth, viability, and for thecorrect assembly of the outer membrane. They represent a defensive barrierwhich helps bacteria to resist to antimicrobial compounds and environmen-tal stresses and are involved in many aspects of host–bacterium interactions asrecognition, adhesion, colonization, and, in the case of extremophile bacte-ria, in the survival under harsh conditions. LPS are also called endotoxinsbecause they are cell-bound and, once released from dead bacteria, play a keyrole in the pathogenesis of Gram-negative infections, in mechanisms asvirulence, tolerance for commensal bacteria, and symbiosis. In mammalianhosts, they trigger the activation of both the innate and the adaptativeimmune system whereas in plants they activate the innate immunity defensesystem, also called basal defense.

LPSs are biosynthesized according to their common structural architec-ture. They are composed of a hydrophilic heteropolysaccharide (formed bycore oligosaccharide and a polysaccharide moiety) covalently linked to alipophilic domain termed lipid A, which is embedded in the outer leaflet ofthe outer membrane and anchors these macromolecules there. The LPSleaflet is stabilized by electrostatic interaction of the negatively charged


groups present in the lipid A and in the core region (phosphate groups,uronic acids) with divalent cations (Mg2þ, Ca2þ) which contribute to linkthe LPS molecules to each other. While a typical glycerophospholipidbilayer is a flexible and fluid system, the LPS layer is a semirigid structurewith a highly ordered structure and low fluidity. Such a highly structuredbarrier is obtained by the rigidity of the lipid A-inner core saccharidebackbone, the tight packing of the fatty acid residues together with thepresence of such strong electrostatic interactions. The low permeability ofthe Gram-negative outer membrane explains their lower susceptibility tohydrophobic molecules and/or negatively charged antibiotic than Gram-positive microorganisms.

The three LPS domains are genetically, biosynthetically, biologically,and chemically distinct. When present, the polysaccharide moiety confers asmooth appearance to the colony on agar plates and in this case, LPS arecalled smooth (S)-form LPS. The polysaccharide can be the O-specificpolysaccharide (OPS, O-antigen), the enterobacterial common antigen(only in Enterobacteriaceae), or a capsular polysaccharide (CPS) as, for exam-ple, colanic acid. The absence of the polysaccharide gives the colony arough aspect and, thus, the LPS is named rough (R) form or, chemicallymore correct, lipooligosaccharide (LOS). R-Form LPS may occur in bothwild and laboratory strains possessing mutations in the genes encoding thepolysaccharide biosynthesis or transfer. It was demonstrated that laboratorydeep-rough mutants are able to survive in vitro, and, thus, it has beenthought for a long time that a short core region linked to lipid A representsthe minimal structure with which bacteria are still viable. However,recently viable mutants were generated which contained only a precursorof LPS biosynthesis (the lipid IVA) in the outer membrane, proving thatunder certain conditions only a lipid A partial structure is required toguarantee outer membrane function and bacterial survival (Meredith et al.,2006). However, in tissues or body fluid, many pathogenic bacteria can onlysurvive expressing the polysaccharide, which protects them from the hostenvironment. Nevertheless, a good number of highly pathogenic Gram-negative bacteria possess an R-form LPS: Neisseria meningitidis, N. gonor-rhoeae, Haemophilus influenzae, Bordetella pertussis, Campylobacter jejuni, andseveral other opportunistic pathogens as Pseudomonas and Burkholderia. Inthese species, the outer core oligosaccharide is often branched, contributesto the viability and to the membrane function, and its relative variability(similar to that of the polysaccharide in S-form LPS) leads to serologicalspecificity.

In this chapter, we describe the protocols for the extraction of bothLPS forms and their chemical characterization, that is, analyses of thecarbohydrate and fatty acid content, their absolute configuration, andtheir attachment sites. Spectroscopical approaches are out of the aim ofthis chapter.


Readers should note that the protocols described in this chapter repre-sent commonly used original referenced protocols, however, there arechanges originating from state-of-art revisions created in our laboratories.

2. LPS and LOS Extraction Procedures

In the course of the analysis of microbial carbohydrate, the firstimportant step represents the extraction procedure in order to isolate thematerial of interest from the biomass in rather pure form.

In particular, the first two protocols, phenol/chloroform/light petro-leum (PCP) and phenol/triethylamine (TEA)/EDTA operate withoutbreaking the cells which instead happens with the third one, the hotphenol/water extraction, which gives good LPS yields together withnucleic acid or protein contaminants.

Consequently, the first two methods have the advantage that lowamounts of nucleic acid and/or proteins are coextracted with the LPS,even though the LPS extraction may be incomplete. Then, in the case ofa new bacterial strain, it is not possible to know in advance the partitionbehavior of its LPS molecules, and therefore applying only the PCP or hotphenol/water extraction may result in no LPS. In addition, it is advisable tooperate two or more extraction protocols in tandem, with the hot phenol/water method placed at the end of the sequence (Fig. 5.1).

The three methodologies proposed yield five extraction phases(Fig. 5.1), which must be carefully checked for carbohydrate material beforebeing discarded.

Finally, LPS yields range from 0.1% to 6% (wLPS/wdry_cells), thereforehigher yields might be related to higher amounts of contaminating substances.

Drycells

PCPSolid

+Supern

SolidPhOH/TEA /

EDTAH2O /PhOH/

65–70 ºC+ +

Supern Phenol phase (+ solid)

Water phase

• LOS precipitation with water • Wash the precipitate with acetone, suspend it in water and freeze dry it• Dialysis of the supernatant and freeze-drying

• Dialysis • NaCl • Water• Freeze-drying

• Dialysis• Removal of the solid (centrifugation)• Freeze-drying

• Dialysis• Freeze-drying

Figure 5.1 Tandem application of the three extraction protocols to a sample. PCP andwater/phenol are the most widely used, but phenol/EDTA/TEA can be insertedamong the other two. Fractions resulting from the freeze-drying are those whichmight contain sample.


2.1. PCP extraction of LOS (Galanos et al., 1969)

This protocol is suitable for LOS isolation and it is advisable to start with drycells which are obtained from wet bacteria by successive extraction/washingwith absolute ethanol, then with acetone (twice) and diethylether (all at20–22 �C, RT), and then drying under a fume hood.

Reagents and equipment

2.1.1 PCP solution (work under a fume hood): mix 90% aqueous phenol,chloroform, and light petroleum in the proportion 2:5:8 (v/v/v).If the solution appears opalescent, gradually add pieces of solidphenol until the solution is clear.

2.1.2 Stirrer2.1.3 Centrifuge2.1.4 Rotary evaporator2.1.5 Freeze-drier

2.2. Methods

2.2.1 Suspend the dry biomass in the PCP solution (�2.5%, w/v) and stirfor 30 min at RT. A blender or an ultra-turrax disperser may be used,but pay attention and avoid the excessive warming of the solution.

2.2.2 Centrifuge the slurry, collect the supernatant in the evaporation flask,and treat twice the pellet as in step 1, collecting the supernatantstogether.

2.2.3 Save the pellet for later extraction(s).2.2.4 Remove the low boiling solvents (chloroform and light petroleum)

from the supernatant by rotary evaporation. Phenol and the watertraces will remain in the evaporation flask.

2.2.5 Transfer the phenol solution in a tube suitable for centrifugation.2.2.6 Add water dropwise to the phenol solution, slowly and mixing every

time, until the LOS has precipitated. Let the sample stand every nowand then for some time. Be careful and avoid phase separation,otherwise any precipitated LOS cannot be sedimented by centrifu-gation since it is present at the phenol/water phase border. In suchcases, the whole sample has to be evaporated again and the procedureof dropwise adding water has to be repeated. If no LOS precipitatesand there is the danger of phase separation, let the sample standovernight, a precipitate may be present next morning.

2.2.7 Centrifuge the precipitate, wash it with 85% aqueous phenol, thenwith acetone (two to three times), let it dry, suspend it in water, andfreeze-dry it.

2.2.8 With regard to the phenol supernatant, dilute it 10 times with water,dialyze it until the phenol smell has disappeared, and freeze-dry it.


2.3. Phenol/TEA/EDTA extraction (Ridley et al., 2000)

This procedure works well either on dry or on wet cells.


2.3.1 Extracting solution: 0.25 M EDTA, 5% phenol, pH 6.9, correctedwith TEA

2.3.2 Dialysis tube, cutoff 12,000–14,0002.3.3 Oven2.3.4 Stirrer2.3.5 Centrifuge2.3.6 Freeze-drier

2.4. Methods

2.4.1 Equilibrate the oven with the stirrer inside and the extracting solu-tion at 37 �C.

2.4.2 Suspend the biomass in the extraction buffer (dry cell 2.5%, wet cells20%) and stir the slurry at 37 �C, 30 min.

2.4.3 Centrifuge, collect the supernatant, and extract the pellet a secondtime.

2.4.4 Save the pellet for further extractions.2.4.5 Combine the two supernatants and dialyze them against 100 mM

NaCl, changing the solution every 3–4 h, at least for six times (seeNotes and tips).

2.4.6 Dialyze against water and recover the sample via freeze-drying.

Notes and tips

Po
int 2.4.5: EDTA is slowly removed during the dialysis process and its presence may cause the turgidity (and the rupture) of the dialysis bag. Theuse of ionic strength (at least 100 mM) facilitates EDTA removal and avoidsthe above-mentioned problems.
2.5. Hot phenol/water extraction (Westphal and Jann, 1965)

This method works equally well on dry or wet cells and it is not selective forLPS or LOS.


2.5.1 90% phenol2.5.2 Dialysis tubes, cutoff 12,000–14,0002.5.3 Oven2.5.4 Stirrer


2.5.5 Refrigerated centrifuge2.5.6 Freeze-drier

Methods

2.5.7 Equilibrate the oven with the stirrer inside and the solutions (waterand 90% phenol) at 65–70 �C.

2.5.8 Suspend the biomass in the warm water and add an equal volume ofphenol (final concentration: dry cell 2.5%, wet cells 20%).

2.5.9 Stir at 65–70 �C for 30 min. Let it cool to RT then.2.5.10 Centrifuge at 4 �C: cell debris will sediment on the bottom and the

solution will divide in two to three areas: the phenol layer (bottom),eventually a milky interphase, and the top (water) layer.

2.5.11 Separate the water layer from the rest, and add an equal volume ofprewarmed water to the remaining material (milky interphase, thephenol phase, and the cell debris).

2.5.12 Repeat the extraction twice, pooling the water supernatants.2.5.13 Dialyze (separate flasks) the water and the phenol phases until the

phenol smell has disappeared and freeze-dry.2.5.14 The exhaust pellet can be saved or discarded.

3. Purification of the Crude Extracts

Mixtures of LPS, LOS, and CPS (if present) are difficult to purify.There is a general protocol to remove nucleic acids and CPS by repeatedultracentrifugation (see below); however, separation of LPS and CPS maynot be obtained. In such cases, it is recommended to remove the (usuallynegatively charged) CPS and the nucleic acids by precipitation with Cetav-lon ( Jones, 1953; Westphal and Jann 1965). However, proteins and nucleicacids may be removed enzymatically, as described in the following. TheLPS or LOS obtained possess a good degree of purity and are ready forchemical characterization (scheme presented in Fig. 5.2).

3.1. Enzymatic hydrolysis


3.1.1 Digestion buffer: 100 mM Tris, 50 mMNaCl, 10 mMMgCl2, bufferat pH 7.5 with 1 M HCl

3.1.2 RNAse solution: 2 mg/ml water3.1.3 DNAse solution 2 mg/ml water3.1.4 Proteinase K solution: 5 mg/ml water

Absolute configuration

(1) Methanolysis(2) n-Hexane extraction

n-Hexane(Top layer)

(Bottom layer)

Methanol

LPS

Methylation analysis(Fig.5.5)

Fatty acid methyl esters

Methylglycosides Acetylation Acetylatedmethylglycosides

GC–MSLipids and

monosaccharidesidentification

(1) Octanolysis(2) Acetylation

GC–MS(Absolute configuration)

GC–MS

Figure 5.2 Convenient strategy for GC-MS chemical analysis of LPS (or LOS) pur-ified samples.


3.1.5 Oven3.1.6 Dialysis tube, cutoff 12,000–14,0003.1.7 Freeze-drier3.1.8 Centrifuge and ultracentrifuge (optional)

3.2. Method

3.2.1 Dissolve the crude extract (50 mg) in the digestion buffer (9 ml).3.2.2 Add RNAse and DNAse solution, 0.5 ml each.3.2.3 Incubate at 37 �C overnight with no stirring.3.2.4 Add 0.1 ml of Proteinase K solution and incubate at 55 �C for 4–5 h.

If necessary, add a second aliquot of Proteinase K solution andprolong the incubation overnight.

3.2.5 Dialyze and freeze-dry the sample.3.2.6 For separation of CPS, dissolve the sample in water at a concentration

2.5 mg/ml and centrifuge at 10,000�g, 4 �C, 30 min (see Notes andtips).

3.2.7 Remove the precipitate (if present) and ultracentrifuge the superna-tant at 300,000–500,000�g, 4 �C, overnight.

3.2.8 Collect the supernatant, suspend the solid in the same amount ofwater originally used, and repeat the ultracentrifuge treatment foranother two times.

3.2.9 Do not combine the three supernatants; analyze them and theprecipitate with the methodologies illustrated in the followingsections.

No


tes and tips

Po
int 3.2.6: Such ultracentrifugation treatment may lead to separation of CPS from LPS or LOS molecules. It is recommended in those cases inwhich the occurrence of CPS is suspected.
4. SDS-PAGE

Detailed protocols for the preparation and run of an electrophoresisgel are available from the instrument manufacturer. This section will coverthe two main staining procedures used for LPS detection and their adapta-tion to anionic polysaccharides.

Performing a typical 12% SDS-PAGE followed by silver staining, LOSmolecules, by virtue of their low molecular mass, migrate faster and arefound almost at the bottom of the gel. The LPS banding pattern is polydis-perse, due to the occurrence of molecules with a different number ofrepeating units in the O-antigen. As a result, LPS bands start (from thetop) in the upper 30% of the gel and end with the LOS band, giving rise tothe so-called ladder-like pattern.

Differently from LPS or LOS, capsular anionic polysaccharides can be seenonly if prestained (or fixed) with alcian blue, and usually are located above theLPS bands, although they can ‘‘invade’’ the LPS area (due to diffusion).

4.1. Kittelberg and Hilbink protocol (Kittelberg andHilbink, 1993)


4.1.1 Rotatory shaker4.1.2 Fixing solution: 40% EtOH, 5% AcOH4.1.3 Oxidizing solution: 0.7% sodium metaperiodate (NaIO4) in the

fixing solution (4.1.2.), 100 ml4.1.4 0.1% Silver nitrate (AgNO3) in water, 100 ml4.1.5 Developing solution: formaldehyde (20 ml) in 100 ml of 3% Na2CO3

4.1.6 Stop solution: AcOH 1% or 7%4.1.7 Farmer solution: 0.3% sodium thiosulfate, 0.15% potassium ferricya-

nide, 0.05% Na2CO3

4.2. Methods

At any step, even if not explicitly mentioned, the gel is gently shaken on arotatory shaker.


4.2.1 Fix the gel with the fixing solution (4.1.2) for at least 1 h.4.2.2 Treat the gel with the oxidizing solution (4.1.3) for 10 min.4.2.3 Wash the gel with water: 3 � 100 ml, 30 min each. If NaIO4 is not

perfectly removed, the gel background may overstain.4.2.4 Treat the gel with silver solution (4.1.4) for 30 min.4.2.5 Wash the gel with water for few seconds.4.2.6 Color development: treat the gel with the formaldehyde solution

(4.1.5). The color should start to appear within a few minutes andshould become more intense as time passes. The gel backgroundmight turn to gray or dark gray. Do not prolong the developmentfor more than 30 min.

4.2.7 Stop the stain development with 1% AcOH (10 min) if the gel hasto be treated with the Farmer solution afterwards. Otherwise, use7% AcOH (10 min).

4.2.8 Wash the gel with water: 3 � 100 ml, 10 min each. The gelcan now be stored, dried, scanned, or treated with Farmer solution.

4.2.9 To optimize staining contrast, treat the gel with Farmer solution(4.1.7) for a few seconds (see Notes and tips).

4.2.10 Stop decoloration with 1% or 7% AcOH if you wish to enhance thesilver staining with a second staining passage.

4.2.11 Wash the gel with water: 3 � 100 ml, 10 min.4.2.12 Store the gel, or restart from point 4.2.4.

Notes and tips

Poin
t 4.2.9: Farmer solution: this solution allows to attenuate silver overstaining, especially that appearing as background. It is important toremember that the background may destain faster depending on the sample;however, excessive time exposure of the gel to the solution might eliminateeverything. The destaining is reversible and the gel can be recovered startingfrom point 4.2.4.
4.3. Tsai and Frasch (1982) protocol


4.3.1 Oxidizing solution: 0.07% aqueous NaIO4 as fixative (150 ml, thesame as 4.1.2)

4.3.2 Silver nitrate solution (150 ml) freshly prepared as follows:142 ml H2O, 0.7 ml 4 M NaOH, 2 ml 25% aqueous NH3, 5 ml20% AgNO3

4.3.3 Developing solution: formaldehyde (80 ml), 8 mg sodium citrate in150 ml H2O


4.4. Method

At any step, even if not explicitly mentioned, the gel must be gently shakenon a rotatory shaker.4.4.1 Fix the gel with the fixing solution (4.1.2) for at least 1 h.4.4.2 Treat the gel with the oxidizing solution (4.3.1) for 5 min.4.4.3 Wash the gel with water: 8 � 100 ml, 10 min each. If NaIO4 is not

perfectly removed, the gel background may overstain.4.4.4 Treat the gel with the silver nitrate solution (4.3.2): 10 min.4.4.5 Wash the gel with water: 4 � 100 ml, 10 min each.4.4.6 Color development: treat the gel with the formaldehyde solution

(4.3.3). The color should start to appear within a few minutes andshould becomemore intense as time passes. Stop the staining with 7%AcOH (10 min) when it reaches the desired intensity.

4.4.7 Wash the gel with water, 3 � 100 ml, 10 min each, and store itas appropriate.

4.5. Detection of acidic polysaccharides(Min and Cowman, 1986)

Reagent

4.5.1 0.05% (w/v) alcian blue in fixing solution (4.1.2)

4.6. Method

4.6.1 Wash the gel with 100 ml fixing solution for 1 h (see Notes and tips).4.6.2 Leave the gel in the alcian blue solution (4.5.1) overnight.4.6.3 Wash the gel with the fixing solution until the background is deco-

lored (see Notes and tips).4.6.4 Proceed with the preferred silver staining procedure.

Notes and tips

Po

Po

int 4.6.1: Removal of SDS from the gel is important or precipitation
of alcian may occur.
int 4.6.3: Alcian blue complexes and fixes anionic polysaccharides
that will appear azure after overnight incubation. The presence of alcianblue does not affect the silver staining but may confer to this materialunexpected colors (usually green).


5. Carbohydrate Analysis: Monosaccharide

Determination, Absolute Configuration, and

Definition of Branching Points

5.1. Monosaccharide determination: Acetylatedmethyl glycosides

The amounts of sample indicated refer to samples extracted from thebiomass without further purification. With the analysis of acetylated methylglycosides it is possible to detect different types of monosaccharides, that is,deoxyhexoses, hexoses, uronic acids, aminosugars, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), neuraminic acid; however, sugars, as fructose and2-deoxy ribose, are completely destroyed.

Each class of monosaccharides (hexoses, deoxyhexoses, etc.) is eluted ina particular range of the chromatogram (see Fig. 5.3), and displays the sameelectronic impact (e.i.) fragmentation pattern (fragmentation of differenttypes of monosaccharides are reported in Fig. 5.4A–F). For a given massspectrum, the peaks most relevant for the recognition of the type of residueare those at high mass values. It must be noted that the molecular ion isnever observed because it is unstable and prone to fragmentation. The mostdiagnostic ion usually observed, even if with low intensity, is the oxoniumion descending from the molecular ion after loss of the anomeric carbonsubstituent, together with the secondary fragments arising from the loss ofacetic acid (60 u), acetic anhydride (120 u), or ketene (42 u) (Lonngren andSvensson, 1974).

ºC220210200190180170160150

Dideoxyhexose

Deoxyhexose

Deoxyhexosamine

Pentose Uronicacid Hexose

Hexosamine

Hexosami-nuronic

acid

252015105 Time (min)

Figure 5.3 Elution order of the monosaccharide as acetylated methyl glycoside deri-vatives in the GC-MS chromatogram with the temperature program: 150 �C 3 min,3 �C/min up to 280 �C, on a SPB-5 column (0.25 mm � 30 m, He as carrier gas). Thedotted gray line indicates the temperature reached at the specific time point of thechromatographic run. Heptoses are found around 27 min, Kdo around 28 min, legio-naminic acid-type residues gives at least a couple of peaks between 32 and 35 min, sialicacid is detected at 38 min.

59 8199115131

153

43

43

43 43

E

C

A

F

D

B

181197

213231255

273 316300 333

375403

40 80 120 160 200 240 280 320

4061

81 98115129145169

183200

215229 243 289 331

80 120 160 200 240 280 320 40 80 120 160 200 240 280 320330

302288228199181156

143127

114

101

8459

43

5774 87102115

129142157

171184

201213

273

242

40

5769

86 102115128

139 157170187 217 259

8060 120100 140 160 180 200 220 240 260 40 8060 120100 140 160 180 200 220 240 260

360 400 40

61

43

83

101 143

124 165 199224242 284

266 302325 362

343 386414 446

80 120 160 200 240 280 300 320 360 400 440

Figure 5.4 Fragmentation pattern of acetylated methyl glycosides prepared fromdifferent types of monosaccharides: (A) pentose, (B) deoxyhexose, (C) hexose, (D)2-aminohexose, (E) Kdo, (F) sialic acid.



5.1.1 Screw cap pyrex tubes with caps lined with inert material (Teflon).Alternatively, pyrex tubes can be sealed by fusing

5.1.2 Anhydrous hydrochloric methanol, 1–1.25 M, n-hexane, dry pyri-dine, and acetic anhydride

5.1.3 Heating block5.1.4 GC-MS equipped with Supelco SPB-5, or SPB-1 column

(0.25 mm � 30 m) or equivalent columns from other manufacturers

5.2. Method

5.2.1 Dry the sample (0.5–1.0 mg of crude product or 0.2 mg of purifiedsample) over a drying agent, under continuous vacuum, for a coupleof hours (see Notes and tips).

5.2.2 Add 1 ml of methanolic hydrochloric acid and close the tube tightly.5.2.3 Incubate the sample at 80 �C overnight (see Notes and tips).5.2.4 Cool the sample and add n-hexane until the two layers are

almost equal.


5.2.5 Collect the hexane phase (top) separately and repeat the extractiontwice, combining all top layers. Follow point 6.2.1.

5.2.6 Dry the methanolic phase in a stream of air, eventually warming itgently (40 �C).

5.2.7 Acetylate the dried methanolic extract with pyridine (200 ml) andAc2O (100 ml), 80 �C, 30 min.

5.2.8 Dry the products in a stream of air. Last traces of pyridine can beremoved by adding few drops of toluene and evaporation.

5.2.9 Dissolve the product in acetone (100 ml) and analyze (1–3 ml) by GC-MS with the temperature program 150 �C 3 min, 3 �C/min up to280 �C, 280 �C 10 min.

Notes and tips

Po
int 5.2.1: Due to the kind of chemical cleavage, each monosaccharide will give rise to different O-methyl glycosides (a- and b-, furanose andpyranose ring) and more than one peak will be observed in thechromatogram.
int 5.2.3: Overnight incubation is necessary for samples of unknown
Pocomposition. If occurrence of 2-amino-2-deoxy-sugars can be ruled out,methanolysis can proceed for 2 h.
5.3. Monosaccharide absolute configuration (octylglycosides, see Notes and tips: Leontein et al., 1978)

The identification of the absolute configuration of a monosaccharide is possibleby the preparation of the appropriate octyl glycoside standard, as shown inFig. 5.5. This approach avoids the use of rare (if available) monosaccharidesand minimizes the use of the (cost-effective) enantiomeric pure alcohol. Therationalebehind theuseof racemic2-octanol is toproduce twodiastereoisomersstarting fromanenantiopuremonosaccharide (Dor L). For instance (seeFig. 5.5),starting from D-Glc, the diastereoisomers D-Glc-(þ)-oct. and D-Glc-(�)-oct.are synthesized, and these two products have the same chromatographic behav-ior of their enantiomers, L-Glc-(�)-oct. and L-Glc-(þ)-oct., respectively. As aresult, analysis and comparisonof the chromatogramsobtained after the reactionof D-Glc with the racemic and the enantiopure 2-octanol yield to the determi-nation of the retention time of D-Glc-(þ)-oct. and L-Glc-(þ)-oct.

Reagents and equipments

5.3.1 Screw cap pyrex tubes with caps lined with inert material (Teflon).Alternatively, pyrex tubes can be fused by heat

5.3.2 Acetyl chloride, pure 2-(þ)-octanol (or the enantiomer), racemic2-(�)-octanol, dry pyridine, and acetic anhydride

5.3.3 Heating block

2-(+)-octanol, H+D-Glc

D-Glc-(+)-oct.

D-Glc-(+)-oct.

Acetyl

GC–MSL-Glc-(–)-oct.

2-(±)-octanol, H+

D-GlcD-Glc-(+)-oct. Acetyl

GC–MSL-Glc-(–)-oct.

D-Glc-(–)-oct.

L-Glc-(+)-oct.

L-Glc-(+)-oct.

Figure 5.5 Strategy used for the construction of the appropriate octyl glycosidestandard. D-glucose is used as an example and L-Glc is not necessary for the constructionof the standard. Reaction of 2-(�)-octanol with D-Glc produces a mixture of diaster-eoisomers, D-Glc-(þ)-oct., and D-Glc-(�)-oct.; the retention time of these compoundsis the same as the corresponding enantiomers, L-Glc-(�)-oct., and L-Glc-(þ)-oct.,respectively (gray tone in the scheme), making the identification of the L-Glc-(þ)-oct. possible without the use of this rare monosaccharide.


5.3.4 GC-MS equipped with Supelco SPB-5, or SPB-1 column(0.25 mm � 30 m) or analog columns from other manufacturers

5.4. Methods

Construction of an octyl glycoside standard necessary for the determinationof the absolute configuration.5.4.1 Prepare two vials, each with the same amount of the reference

compound (0.2 mg).5.4.2 Dry the samples over a drying agent, under continuous vacuum, for

a couple of hours.5.4.3 Add to one tube 100 ml 2-(þ)-octanol, and to the other tube the

same amount of the racemic alcohol.5.4.4 Add to each tube 15 ml of acetyl chloride: pipette the liquid dipping

the tip in the octanol solution.5.4.5 Close the vials and incubate overnight at 60 �C5.4.6 Remove the solvent in a stream of air warming at 40 �C.5.4.7 Acetylate with pyridine (200 ml) and Ac2O (100 ml), 80 �C, 30 min.5.4.8 Dry the products in a stream of air. Last traces of pyridine can be

removed by evaporation after adding a few drops of toluene.5.4.9 Dissolve the product in acetone (200 ml) and analyze it (1 ml) via

GC-MS with the temperature program: 150 �C 3 min, 3 �C/minup to 280 �C, 280 �C 10 min.


5.5. Determination of the absolute configuration ofcarbohydrates in a sample

5.5.1 In this case it is necessary to have only one vial containing the sample.It is possible to start from polysaccharide material, or better, from theacetylated methyl glycoside.

5.5.2 When starting with the pure polysaccharide, the sample is treated asfrom point 5.4.2. When starting from the acetylated methyl glyco-sides, then this step is skipped.

5.5.3 Prepare the 2-(þ)-octanol derivative as described in 5.4.3.5.5.4 Dissolve the sample in 100 ml chloroform and inject 1 ml via GC-MS.

Use the reference compound for the appropriate assignment.

Notes and tips

Po
int 5.3 The diastereoisomers obtained with glucosamine and 2-(�)- octanol are hardly separated by GC-MS, whereas if 2-butanol is usedinstead, it works (Gerwig et al., 1978).
5.6. Determination of monosaccharides branching points(methylation analysis): protocol for neutral (Ciucanu andKerek, 1984) and uronic acid containing polysaccharides

The determination of the substitution pattern of one monosaccharideresidue in a polysaccharide is possible by analyzing partially methylatedalditol acetates (AAPM) that result after a series of reactions (Fig. 5.6).Basically, the methylation reaction transforms the available free hydroxylfunctions of the polysaccharide into methyl ethers (Fig. 5.6, methylationstep), N-acyl aminosugars are N-methylated as well. Uronic acids areesterified and the methyl ester function needs to be reduced before thehydrolysis step, otherwise the residue cannot be detected. The reductionreaction with NaBD4 transforms the methyl ester function in a hydro-xymethyl group with two deuterium atoms (Fig. 5.6, ester reductionstep). The hydrolysis step cleaves all the glycosidic linkages and partiallymethylated monosaccharides are released, the free hydroxyl groups thatnow appear are those originally involved in glycosidic linkages (Fig. 5.6,hydrolysis step). Successively, the anomeric position is marked with onedeuterium atom using NaBD4 reduction, and two new hydroxyl func-tions appear originating from ring opening (Fig. 5.6, reduction). Acety-lation of these compounds yields to the so-called AAPM. The molecularion is never detected in the spectrum, and interpretation of GC-MSfragmentations follows few rules (given below), leading to the localizationof the methyl and acetyl groups on the alditol backbone (examples inFig. 5.7).

HOH2C HOOC6 6

5 5O O

O

O

O O

OO O

AAPM

1

2

3

4

5

6

O

Methylation

Hydrolysis

Acetylation

O OHO HO

OH

4 4

3 2 1 OH3 2 1

H3COH2C H3COOC

H3CO

H3CO

H3CO

H3CO

H3COH2C

CH2OCH3 CD2OH CH2OCH3 CD2OAc

H3COH2C

HOD2C

HOD2CH3CO

H3CO H3CO

HO

HO OH

OH

OCH3

OCH3

OCH3 OCH3OCH3

OCH3

H

OH

OH

CHDOH

H

H

H

H3CO

OCH3

H

OH

OH

H

H

H

H3CO

OCH3

H

OAc

OAc

H

H

H

H3CO

OCH3

H

OAc

OAc

H

H

H

CHDOH CHDOAc CHDOAc

Esterreduction(NaBD4)

Reduction(NaBD4)

OCH3

OO

OO

O

Figure 5.6 Derivatization protocol used to determine the substitution pattern of themonosaccharide residues of a polysaccharide. Carbon atoms of the monosaccharide arenumbered and the same numbers are used for the corresponding hydroxyl functions,protons are omitted for clarity. At the end of the reaction sequence, the monosaccharidederivative detected is a partially methylated and acetylated alditol (AAPM). O-Methylgroups (at C-2, C-3, and C-6) indicate free hydroxyl function of the original polysac-charide; O-acetyl groups position is related to the substitution point (C-4) and on thetype of the ring closure (pyranosidic, C-1 and C-5) of the monosaccharide. C-6 of auronic residue is doubly deuterated and acetoxylated.

277118

162

118

162

159

203

A B C

233

307

263

318

233

CHDOAc

CH2OCH3 CD2OAc CH2OCH3

OCH3

H

OAc

OAc

H

H3CO

H

H

CHDOAc CHDOAc

OCH3

H

OAc

OAc

H H

H3CO

H

H

H

NCH3

Ac

OAc

OAc

H3CO

H

H

Figure 5.7 Ions expected from the primary fragmentation of the AAPM descendingfrom: (A) 4-substituted glucose, (B) 4-substituted glucuronic acid, (C) 4-substituedN-acetyl glucosamine



– Primary fragments are originating from the carbon–carbon bond cleavageof the alditol backbone.

– Intensities of the primary fragments decreasewith increasingmolecularmass.– The (positive) charge is placed on the methoxylated (or on the nitrogenbearing) carbon.

– Fission between two methoxylated carbons is favored with respect to anacetoxylated and a methoxylated carbon, in turn favored with respect tothat between two acetoxylated carbons.

– For neutral and uronic descending alditols, primary fragments containingC-1 are even numbers, whereas those containing C-6 are odd numbers,the reverse is true for amino sugars when the fragment contains thenitrogen atom.

– Secondary fragments are formed from the primary ones by single orconsecutive eliminations of formaldehyde (30 u), methanol (32 u),ketene (42 u), acetic acid (60 u), and methyl acetate (74 u).

– Elimination of methanol is observed when methoxyl group is situated atthe b-position to the carbon having the formal charge, the same happensfor acetoxyl group elimination.


5.6.1 Screw cap pyrex tubes with caps lined with inert material (Teflon)5.6.2 Dry DMSO, NaOH pellets, pure CH3I, CHCl3, 2 M trifluoroacetic

acid, NaBD4, pyridine, Ac2O, EtOH, MeOH, i-PrOH, glacialAcOH, 1 M HCl

5.6.3 Stirrer and stirring bars appropriate for the pyrex tube5.6.4 Ultrasound bath5.6.5 Centrifuge5.6.6 Heating block5.6.7 GC-MS equipped with Supelco SPB-5, or SPB-1 column

(0.25 mm � 30 m) or analog columns from other manufacturers

5.7. Methods

5.7.1 Dry the sample (0.5–1.0 mg of a purified product) together with thestirring bar over a drying agent, if possible in a thermoregulateddesiccator, overnight.

5.7.2 From now on, work under a fume hood. Dissolve the sample in�1 ml of dry DMSO (see Notes and tips).

5.7.3 Pulverize in a pestle 2–3 NaOH pellets and add the content of thespun of a small spatula (�100 mg) to the solution (see Notesand tips).


5.7.4 Seal the sample and stir the solution for 2 h (see Notes and tips). Atthe end of this time perform one last sonication cycle (5 min).

5.7.5 Freeze the solution in an ice bath and add 200 ml of CH3I (see Notesand tips).

5.7.6 Leave the solution stirring for at least 2 h (if necessary, overnight)after DMSO has melted.

5.7.7 Apply a gentle stream of air for 1 h to remove CH3I.5.7.8 Extract the solution with water (10–15 ml) and CHCl3 (1–2 ml),

five times, replacing the top layer with water, each time. Phaseseparation is better achieved if the solution is spun in a centrifuge.

5.7.9 Dry the organic phase (see Notes and tips).5.7.10 If the sample contains uronic acids, follow the next steps, otherwise

go to point 5.7.15.5.7.11 Dissolve the sample in 50%MeOH solution in water (1 ml) and add

the tip of a small spun of NaBD4 (�5 mg). Stir at 4 �C, overnight.5.7.12 Destroy the NaBD4 excess with one to two drops of 1 M HCl and

dry the solution in a stream of air, eventually warming at 40 �C.5.7.13 Add 300 ml of MeOH and one drop of acetic acid and dry by

warming at 40 �C. Repeat three times.5.7.14 Add 300 ml of MeOH and dry by warming at 40 �C. Repeat

three times.5.7.15 Hydrolyze the sample with 2 M trifluoroacetic acid (200 ml) at

120 �C, 1 h (see Notes and tips).5.7.16 Dry the sample in a stream of air, adding few drops of i-PrOH. Do

not warm: partially methylated monosaccharides produced afterhydrolysis are volatile.

5.7.17 Once the sample is dried, repeat the evaporation process withi-PrOH only for three times. This treatment is necessary to removetrifluoroacetic acid traces.

5.7.18 For reduction, add to the sample the tip of a small spun of NaBD4

(�5 mg) and 200 ml of EtOH. Keep the sample capped, at RT for atleast 1 h.

5.7.19 Destroy the NaBD4 excess with one to two drops of glacial AcOH,and dry the solution in a stream of air.

5.7.20 Add 300 ml of MeOH and one drop of acetic acid and dry in astream of air, without warming.

5.7.21 Add 300 ml of MeOH and dry without warming. Repeatthree times.

5.7.22 Keep the sample in a desiccator, without vacuum, for 1 h.5.7.23 Acetylate with Pyr (200 ml) and Ac2O (100 ml), at 80 �C for 30 min.5.7.24 Dry in a stream of air and add with water (4 ml) and CHCl3

(1–2 ml).5.7.25 Extract three times with CHCl3, replacing each time the water phase.

Use of a centrifuge to separate the two phases is recommended.


5.7.26 Dry the organic phase, dissolve it in 50–100ml of acetone and analyze1–3 ml by GC-MS under the conditions indicated for the analysis ofacetylated methyl glycosides. Please note that AAPM are volatile, donot exceed in drying the sample more than necessary.

Notes and tips

Poin

Poin

Poin

Poin

Poin

t 5.7.2: Polysaccharides are not very soluble in DMSO, therefore
sample dispersion is important for the outcome of the whole procedure.Alternate sonication and stirring to reach the best dispersal of the sample.
t 5.7.3: NaOH is hygroscopic and adsorbs both humidity and
carbonic anhydride. When pulverizing it, work quickly and avoid toweigh the powder which would spoil your reactive. NaOH in DMSO ispartly soluble, for better results stir and sonicate the solution after addition ofthe solid. A good result is obtained when the solution is opalescent. If astrong excess of NaOH is used, then a precipitate is observed, even thoughthis usually does not affect the reaction outcome. In this methodology,NaOH in DMSO acts as both a dryer (for the water traces) and as a base (todeprotonate the monosaccharide hydroxyl functions).
t 5.7.4: The time contact between the sample and the NaOH is
Poinsomehow dependent on the solubility of the sample in DMSO. If it issoluble, or if a good dispersion of the polysaccharide is reached easily, thenstirring the solution for a couple of hours is usually enough; otherwise, it is agood practice to prolong the contact time between the reactives.
t 5.7.5: CH3I and NaOH react together and the reaction is rather
exothermic. It seems that a good result is reached when the reactionproceeds slowly, so that the CH3I contact with the DMSO solution isgradual and dictated from the melting speed of the frozen solution.
t 5.7.9: If after drying, the organic phase appears deliquescent, it
means that it contains still some traces of DMSO; extract it again.
t 5.7.15: Hydrolysis time may need some optimization. This
condition is rather general, but amino hexoses may be underestimated.Alternatively, prolong hydrolysis time to 2 h or use 4 M trifluoroaceticacid at 100 �C for 4 h. In this last case hydrolysis is rather complete andmonosaccharides degradation is low.
6. Fatty Acids Compositional Analysis (GC-MS)

6.1. Total fatty acid composition by methanolysis

Fatty acids occurring in LPS can be divided in essentially three differentclasses: (1) simple and saturated fatty acids, as 14:0; (2) C-3 hydroxylatedfatty acids, as 14:0(3-OH); and (3) C-2 hydroxylated fatty acid. According


to the formalism adopted, ‘‘14’’ identifies a 14-carbon atom fatty acid, ‘‘:0’’indicates that the residue is saturated (without double bonds), ‘‘2-OH’’ (or3-OH) indicates that a hydroxyl function occurs at carbon 2 (or 3) of themolecule.

The assignment of different fatty acid methyl esters is possible bycomparison of their retention time with those of commercially availablestandards (Fig. 5.8) and by analysis of their fragmentation pattern. Ingeneral, e.i. spectra of the methylester derivatives are rather informativeand the molecular ion (M) is usually visible for type 1 and 2 fatty acids,although its intensity is rather low. For type 1 fatty acids, the spectrum(Fig. 5.9A) is dominated from the fragment at 74 u originated fromMcLafferty (ML) rearrangement, which is produced from the fission ofthe Cb–Cg linkage, together with the extraction of a g-proton, the proton-ation of the carbonyl oxygen, and the formation of a radical cation. Most ofthe other ions differ for 14 u and descend from carbon–carbon linkageruptures. The series containing the methylester group is predominant.

With regard to type 2 lipids (Fig. 5.9B), identification of the substitutionpattern is possible observing the ML ion at 90 u, which indicates thehydroxylation at C-2. In addition, when the molecular ion is not detected,the rather intense ion at M-59 u (where 59 originates from the carbox-ymethyl function) indicates the length of the hydrophobic tail of thecompound. The other fragmentations observed for this type of lipid areless indicative to define its structure.

350001

2

34

9 1017

5 67 8 1112

13 14 15 16

18

192021 22 23 24

30000

25000

20000

15000

10000

5000

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

Figure 5.8 GC-MS chromatogram of fatty acid methyl ester derivatives commerciallyavailable. GC-MS operative conditions are those listed for acetylated methylglycosides.Compound list: (1) C12:0, (2) C13:0, (3) C12:0(2-OH), (4) C12:0(3-OH), (5) C14:0,(6) i-C15:0, (7) a-C15:0, (8) C15:0, (9) C14:0(2-OH), (10) C14:0(3-OH), (11) i-C16:0,(12) 16:19, (13) C16:0, (14) i-C17:0, (15) 17:0D, (16) C17:0, (17) C16:0(2-OH),(18) C18:29,12, (19) C18:19 cis, (20) C18:19 trans þ C18:111 (21) C18:0, (22) C19:0D,(23) C19:0, (24) C20:0. i- and a- indicate the iso- or anteiso-terminal type of the aliphaticchain of the fatty acid, respectively; D indicates the presence of a ciclopropane ring;‘‘:19’’ means that the fatty acid possesses one unsaturation at C-9.

8000A

7000

6000

5000

4000

3000

2000

1000

0

900B

800700600500400300200100

0

2400

C

2200200018001600140012001000800600400200

040 60 80 100 120 140 160 180 200

40 60 80 100 120

103

140 160 180 200 220 240

40

4355

87

74

97 115 129

143

157 171 185

199211 242

43

55 69 83

97

ML90

ML

111

60 80 100 120 140 160 180 200 220 240

260

183

M-92166

M-59199

M-59

M-31

M

M-50208141123111

125145

156 213

M258

9683

ML74

67

55

43

Figure 5.9 Electronic impact MS spectra of three fatty acid methyl esters representativeof the different types of lipids occurring in LPS (or LOS): (A) C14:0, (B) C14:0(2-OH),(C)C14:0(3-OH).M ¼ molecular ion,ML ¼ fragment fromMcLafferty rearrangement.


Differently from type 1 and 2, type 3 lipid fragmentation pattern(Fig. 5.9C) is dominated by the ion at 103 u: this ion is originating fromthe Cb–Cg fission and it contains the first three carbon atoms of the fatty acidand the positive charge is stabilized from the b-hydroxyl function. Themolecular ion is usually not visible, but an indication of the length of the tail


is obtained observing the ions at M-50 (loss of water and methanol) andM-92 (loss of water, methanol, and ketene). The other fragmentationsobserved for this type of lipid are less indicative to define its structure.

Please note that hydroxylated fatty acid methyl esters might tail on theGC column, up to be lost in baseline. This behavior is observed when thecolumn is not performing correctly, for example, it is an aged one.



6.1.2 Anhydrous hydrochloric methanol (1–1.25 M), n-hexane6.1.3 Heating block6.1.4 GC-MS equipped with Supelco SPB-5, or SPB-1 column


6.2. Methods

6.2.1 Follow the protocol from point 5.2.1 to 5.2.5.6.2.2 Dry the combined n-hexane phases in a stream of air. Please note that

lipid methylesters are volatile, do not exceed in drying the samplemore than the necessary.

6.2.3 Dissolve the product in n-hexane (100 ml) and analyze it (1-3 ml) viaGC-MS with the temperature program: 150 �C 3 min, 3 �C/min upto 280 �C, 280 �C 10 min.

6.3. O-Linked fatty acid



6.3.2 0.5 M NaOH, 1–0.5 M HCl, chloroform, diazomethane6.3.3 Heating block6.3.4 Centrifuge6.3.5 GC-MS equipped with Supelco SPB-5, or SPB-1 column


6.4. Methods

6.4.1 Dissolve the sample (2 mg) in 0.5 M NaOH (1 ml).6.4.2 Incubate the sample at 37 �C for 2 h.


6.4.3 Add HCl to the solution to reach a pH � 3.6.4.4 Extract twice the solution with chloroform, pool the organic phases,

and dry them in a stream of air.6.4.5 Treat the dry organic phase with diazomethane, 0.3–0.4 ml for

15 min or until the solution destains. If destaining is too fast, drythe solution and repeat the treatment.

6.4.6 Dry the solution in a stream of air, dissolve the product in hexane(100 ml) and analyze it (1–3 ml) via GC-MS with the temperatureprogram 150 �C 3 min, 3 �C/min up to 280 �C, 280 �C 10 min.

6.5. Absolute configuration determination of hydroxyl fattyacids (Rietschel, 1976)



6.5.2 4 M HCl, 8 M HCl, 1 M NaOH, 5 M NaOH, dry NaOH, CHCl3,methyl iodide, DMSO, thionylchloride, D- and L-phenylethylamine,pyridine

6.5.3 Heating block6.5.4 GC-MS

6.6. Methods

Treat the sample [0.5–1.0 mg of LPS or 0.1 mg of 14:0(3-OH)] as follows,or use the methylesters from methanolysis starting from point 6.6.6.6.6.1 4 M HCl (200 ml), 2 h, 100 �C.6.6.2 5 M NaOH (200 ml), 30 min, 100 �C.6.6.3 Acidify with 8 M HCl (200 ml).6.6.4 Extract three times with 0.5 ml of CHCl3 and combine all the three

extracts in a vial, dry with air.6.6.5 Dissolve in CHCl3 and treat the sample twice with diazomethane

(under a fume hood), dry the sample.6.6.6 Dissolve the methyl esters with in 1 ml of DMSO.6.6.7 Add a spatula tip of pulverized NaOH.6.6.8 Add 0.5 ml methyl iodide and stir for 30–60 min.6.6.9 Cautiously add water, 5–10 ml.6.6.10 Extract three times with 1 ml of CHCl3, combine the extracts.6.6.11 Wash the CHCl3 phase one time with 50 ml of water.6.6.12 Remove the CHCl3 in a stream of air.


6.6.13 Add 100 ml of 1MNaOH (in MeOH/H2O 1/1, v/v), and heat for1 h at 85 �C.

6.6.14 Add 50 ml of 4 M HCl and 400 ml of H2O.6.6.15 Extract three times with CHCl3 and dry with a stream of air.6.6.16 Add 50 ml of thionylchloride and incubate 10 min at 85 �C.6.6.17 Dry in a stream of air.6.6.18 Add D- or L-phenylethylamine (0.5 ml/100 ml pyridine, without

NaOH).6.6.19 Add 50 ml of this mixture to the sample, incubate 20 min at 85 �C

and dry in a stream of air.6.6.20 Add 600 ml of 1 M HCl and extract twice with 600 ml of hexane.6.6.21 Separate and dry the organic phase, dry, dissolve it inCHCl3, analyze

via GC-MS 0.2/500 ml of sample in the conditions mentioned forfatty acidmethyl esters. Note thatR-configured fatty acids elute first.

REFERENCES

Alexander, C., and Rietschel, E. T. (2001). Bacterial lipopolysaccharides and innate immu-nity. J. Endotoxin Res. 7, 167–202.

Ciucanu, I., and Kerek, F. (1984). A simple and rapid method for the permethylation ofcarbohydrates. Carbohydr. Res. 131, 209–217.

Galanos, C., Luderitz, O., and Westphal, O. (1969). A new method for the extraction of Rlipopolysaccharides. Eur. J. Biochem. 9, 245–249.

Gerwig, G. J., Kamerling, J. P., and Vliegenthart, J. F. G. (1978). Determination of theD and L configuration of neutral monosaccharides by high-resolution capillary g.l.c.Carbohydr. Res. 62, 349–357.

Jones, A. S. (1953). The isolation of bacterial nucleic acids using cetyltrimethylammoniumbromide (Cetavlon). Biochem. Biophys. Acta 10, 607–612.

Kittelberg, R., and Hilbink, F. J. (1993). Sensitive silver-staining detection of bacteriallipopolysaccharides in polyacrylamide gels. Biochem. Biophys. Methods 26, 81–86.

Leontein, K., Lindberg, B., and Lonngren, J. (1978). Assignment of absolute configurationof sugars by g.l.c. of their acetylated glycosides formed from chiral alcohols. Carbohydr.Res. 62, 359–362.

Lonngren, J., and Svensson, S. (1974). Mass spectrometry in structural analysis of naturalcarbohydrates. Adv. Carbohydr. Chem. Biochem. 29, 41–106.

Meredith, T. C., Aggarwal, P.,Mamat, U., Lindner, B., andWoodard, R.W. (2006). Redefin-ing the requisite lipopolysaccharides structure in Escherichia coli. ACS Chem. Biol. 1, 33–42.

Min, H., and Cowman, M. K. (1986). Combined alcian blue and silver staining of glyco-saminoglycans in polyacrylaminde gels: Application to electrophoretic analysis of molec-ular weight distribution. Anal. Biochem. 155, 275–285.

Raetz, C. R., andWhitfield, C. (2002). Lipopolysaccharide endotoxins.Annu. Rev. Biochem.71, 635–700.

Ridley, B. L., Jeyaretnam, B. S., andCarlson,R.W. (2000). Sensitive silver-staining detection ofbacterial lipopolysaccharides in polyacrylamide gels.Glycobiology 10, 1013–1023.

Rietschel, E. T. (1976). Absolute configuration of 3-Hydroxy fatty acid present in lipopo-lysaccharides from various bacterial groups. Eur. J. Biochem. 64, 423–428.


Silipo, A., Erbs, G., Shinya, T., Dow, J. M., Parrilli, M., Lanzetta, R., Shibuya, N.,Newman, M.-A., and Molinaro, A. (2010). Glyco-conjugates as elicitors or suppressorsof plant innate immunity. Glycobiology 20, 406–419.

Tsai, C. M., and Frasch, C. E. (1982). A sensitive silver stain for detecting lipopolysacchar-ides in polyacrylamide gels. Anal. Biochem. 119, 115–119.

Westphal, O., and Jann, K. (1965). Bacterial lipopolysaccharides extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5, 83–91.

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