Monosaccharide Composition of Mucin 163
2.5. HPAEC-PAD
All eluents and chemical products must be of the highest purity available.
1. Gradient pump module (Dionex Bio-LC apparatus, Sunnyvale, CA).
2. A model PAD-2 detector equipped with a gold working electrode. The following pulse
potentials and durations are used for detection: E1 = 0.05 V (t
1
= 360 ms); E2 = 0.70 V (t
2
= 120 ms); E3 = –0.50 V (t
3
= 300 ms) The response time is set to 3 s.
3. Eluent Degas module to sparge and pressurize the eluents with helium (Dionex).
4. Postcolumn with a DQP-1 single-piston pump (Dionex).
5. CarboPac PA-1 column (4 × 250 mm) (Dionex).
6. CarboPac PA-1 guard (4 × 50 mm) (Dionex).
7. CarboPac MA-1 column (4 × 250 mm) (Dionex).
8. CarboPac MA-1 guard (4 × 50 mm) (Dionex).
9. 18 M Ω deionized water (Milli-Q Plus System, Millipore, Bedford, MA).
10. NaOH 50% solution with less than 0.1% sodium carbonate (Baker, Deventer, The Netherlands).
11. Anhydrous sodium acetate (Merck).
12. Acetic acid (glacial, HPLC grade; Merck).
13. Eluents containing sodium acetate should be filtered through 0.45-µm nylon filters
(Millipore) prior to use.
14. Solvents for separation of neutral monosaccharides, hexosamines, and uronic acids (see
Subheading 3.3.3.2.).
a. Eluent 1: Deionized water.
b. Eluent 2: 25 mM NaOH and 0.25 mM sodium acetate.
c. Eluent 3: 200 mM NaOH and 300 mM sodium acetate.
d. Eluent 4: 125 mM NaOH and 10 mM sodium acetate.
15. Solvents for HPAEC separation of sialic acids (see Subheading 3.3.3.3.).
a. Eluent 1: Deionized water.
b. Eluent 2: 5 mM NaOAc.
c. Eluent 3: 5 mM acetic acid (glacial, HPLC grade; Merck).
16. Solvents for separation of a mixture of unreduced and reduced monosaccharides (see
Subheading 3.3.3.4.).
a. Eluent 1: Deionized water.
b. Eluent 2: 1.0 M NaOH.
17. Neu5Ac and Neu5Gc acid (Sigma).
18. A mixture of sialic acids released from bovine submaxillary gland mucin (BSM) (Sigma)
(see Subheding 3.1.1.).
2.6. Electrophoretic Separation of Monosaccharides
1. Capillary zone electrophoresis apparatus fitted with a UV detector (Beckman).
2. Capillary tube (50 µm id × 65 cm) (Beckman). A part of the polyimine coating on the
capillary tube is removed by burning at a distance of 15 cm from the cathode, to allow UV
detection.
3. 2-Aminoacridone (AMAC) (Lambda Fluoreszentechnologie GmbH, Graz, Austria) made
up to 0.1 M in acetic acid:dimethylsulfoxide (DMSO, Acros Organics, Sunnyvale, CA)
(3:17 v/v). The solution is stored at –70°C.
4. 1 M sodium cyanoborohydride (Merck) in water. This solution is made fresh for each
experiment.
164 Michalski and Capon
3. Methods
3.1. Release and Identification of Sialic Acids
Figure 2 illustrates the separation of the different sialic acid species obtained after
hydrolysis of BSM. The different sialic acids may be characterized according to their
specific retention times. Additionally, each sialic acid may be characterized by MS
analysis (8).
3.1.1. Chemical Hydrolysis of Sialic Acids
1. Suspend 1–10 mg of mucins in 5 mL of 2 M acetic acid in a Teflon-capped reaction tube.
2. Hydrolyze for 5 h at 80°C.
3. Dialyze the solution for 24 h against 20 vol of water (1000 mol wt cutoff tubing).
4. Lyophilize the diffusate. Direct analysis can be made at this stage.
5. Further purify sialic acids as follows:
a. Redissolve the dialysate in 1 mL of water.
b. Load the sample on a Dowex AG 50W × 8 (H
+
) (Bio-Rad) column (10 mL).
c. Wash the column with 100 mL of water.
d. Lyophilize the effluent.
e. Resuspend the lyophilysate in 1 mL of water.
Fig. 2. HPLC separation of sialic acid quinoxalinones obtained after mild acid hydrolysis of
BSM. Ac, acetyl; Lt, lactyl; Gc, glycolyl.
Monosaccharide Composition of Mucin 165
f. Load the sample on a Dowex AG 3 × 4A (HCOO
–
) (Bio-Rad) column (1 mL).
g. Wash the column successively with 7 mL of 10 mM , 7 mL of 1 M and 7 mL of 5 M
formic acid.
h. Pool the fractions and lyophilize.
3.1.2. Enzymatic Release of Sialic Acid
1. Resuspend 1–10 mg of mucin in 2 mL of 100 mM HEPES-KOH, pH 7.0, 150 mM NaCl,
0.5 mM MgCl
2
, and 0.1 mM CaCl
2
.
2. Add 200 mU/mL of V. cholerae or 40 mU/mL C. perfringens enzyme.
3. Introduce the solution in a dialysis tube (1000 mol wt cutoff) and dialyze against 5 mL of
the same solvent at 37°C overnight.
4. Collect the filtrate and purify the sialic acid as in Subheading 3.1.1.
3.1.3. TBA-HPLC Quantification of Sialic Acids
3.1.3.1. TBA R
EACTION
1. Sialic acids are released from mucins by mild acid hydrolysis as described in Subhead-
ing 3.1.1. The TBA assay is performed essentially according to Warren (23).
2. Place 40 µL of free sialic acid solution (10–100 pmol/100 µL in water) in an Eppendorf tube.
3. Add 20 µL of sodium periodate (128 mg of sodium metaperiodate, 1.7 mL of phosphoric
acid, and 1.3 mL of water).
4. After 20 min at room temperature, add slowly 0.1 mL of 10% sodium arsenite in 0.1 N
H
2
SO
4,
and 0.5 M Na
2
SO
4
.
5. When the solution appears yellow-brown, gently vortex the tubes.
6. Add 0.6 mL of 0.6% TBA (0.6 g of TBA [Sigma] in 0.5 M Na
2
SO
4
[Merck]).
7. After mixing, cap the tubes and heat at 100°C for 15 min.
8. Chill the tubes on ice and centrifuge before HPLC analysis.
3.1.3.2. HPLC A
NALYSIS
(F
IG
. 3)
1. Equilibrate the column in the working solution.
2. Elute in the isocratic mode at a flow rate of 1 mL/min.
3. Run UV detection at 549 nm.
4. Quantify the sialic acid by integrating the surface of the sialic acid. Obtain the chromophore
peak calibration curve with pure sialic acid solution (1–5 µg of sialic acid in 40 µL of water).
5. Wash the column extensively with 50% acetonitrile in water after use.
3.1.4. Characterization and Quantification of Sialic Acids by HPLC
3.1.4.1. D
ERIVATIZATION WITH
DMB
1. Heat sialic acid samples released by mild hydrolysis in 7 mM DMB, 0.75 M β-
mercaptoethanol, and 18 mM sodium hydrosulfite in 1.4 M acetic acid (100–200 µL) for
2.5 h in the dark.
2. Inject 10 µL of the reaction mixture on the C18 column.
3.1.4.2. E
LUTION BY
HPLC
1. Equilibrate the column in 65% solvent A–35% solvent B.
2. Elute using a linear gradient from 65% A/35% B to 100% B over 60 min followed by
isocratic elution by 100% B for 10 min at a flow rate of 1 mL/min.
3. Achieve on-line fluorescent detection at an emission wavelength of 448 nm and excita-
tion wavelength of 373 nm with a response time of 0.5 s.
166 Michalski and Capon
3.2. Analysis of Monosaccharides by GLC
Complete hydrolysis of oligosaccharide chains may be obtained using concentrated
acid solutions.
3.2.1. Trifluoroacetic Hydrolysis
1. Dissolve the oligosaccharide-alditol sample or the native glycoprotein in 0.5 mL of a 4 M
solution of trifluoroacetic acid (TFA) or a mixture of formic acid:water:TFA (3:2:1 v/v/v).
2. Heat at 100°C for 4 h in Teflon-capped tubes.
3. After hydrolysis, remove the acid by repeated evaporation under reduced pressure. Evapo-
ration is completed by the addition of ethanol.
3.2.2. Formic Acid–Sulfuric Acid Hydrolysis
1. Dissolve oligosaccharide-alditols or native glycoproteins in 0.5 mL of 50% aqueous for-
mic acid and hydrolyze for 5 h at 100°C in a Teflon-capped tube.
2. Repeat step 1 using 0.25 M aqueous sulfuric acid for 18 h at 100°C.
3. Neutralize the hydrolysate with barium carbonate powder, filter, and concentrate to dry-
ness (see Note 1).
3.2.3. Methanolysis
Methanolysis is a widely used method for hydrolysis of both oligosaccharides and
native glycoproteins.
Fig. 3. HPLC analysis of TBA chromophores. 2, NeuAc chromophore; 3, deoxyhexose chro-
mophore.
Monosaccharide Composition of Mucin 167
3.2.3.1. P
REPARATION OF
M
ETHANOL
–HC
L
R
EAGENT
1. Obtain anhydrous methanol by refluxing with magnesium turnings (1 h) followed by dis-
tillation in a dry all-glass apparatus.
2. Generate gaseous HCl by slow addition (10–20 drops/min) of sulfuric acid to 250 g of
solid NaCl.
3. Dry the hydrogen chloride gas through moisture traps containing concentrated sulfuric acid.
4. Then bubble hydrogen chloride gas through anhydrous methanol for 3 to 4 h.
5. Standardize the methanol–HCl reagent to 0.5 M by titration with NaOH and dilution with
anhydrous methanol (see Note 2).
3.2.3.2. M
ETHANOLYSIS OF
O
LIGOSACCHARIDE OR
M
UCUS
G
LYCOPROTEIN
(24)
1. Freeze-dry carefully in Teflon-capped tubes (complete dehydration of samples is the
main condition of success) amounts of glycoproteins or oligosaccharides corresponding
to 10 µg of total sugar to which 1 µg of mesoinositol is added as an internal standard.
2. Add 0.5 mL of methanol–HCl reagent.
3. Heat at 80°C for 24 h.
3.2.4. GLC Analysis of Monosaccharides as Their Methylglycoside
(25)
Trimethylsilylated Derivatives
GLC analysis allows the determination of the monosaccharide composition of gly-
cans with amounts of total sugars not exceeding 1 µg (15–20 pmol of glycoproteins). It
consists of the methanolysis of previously purified glycoprotein, followed by a re-N-
acetylation and a trimethylsilylation leading to trimethylsilylation-derivatives.
3.2.4.1. M
ETHANOLYSIS AND
D
ERIVATIZATION
1. Mix amounts of purified glycoproteins corresponding to 0.5 µg of total sugars with 200 µL
of 0.5 M methanol-HCl mixture for 24 h at 80°C.
2. After cooling the tube, neutralize the acidic solution by adding silver carbonate to give a
pH of 6.0-7.0 as controlled with pH paper.
3. Re-N-acetylate by adding 10 µL of acetic anhydride and keep overnight at room tem-
perature.
4. Centrifuge at 2000g for 5 min and collect the supernatant.
5. To eliminate fatty acid methyl esters, wash the methanolic phase two times with 200 µL
of heptane (remove the upper phase).
6. Dry the methanolic lower phase under a stream of nitrogen.
7. Trimethylsilylate with 20 µL of BSTFA in the presence of 10 µL of pyridine for 1 h at
room temperature.
8. Apply 1–5 µL of the solution of trimethylsilylated methylglycosides to GLC.
3.2.4.2. G
AS
C
HROMATOGRAPHY
C
ONDITIONS
A typical GLC chromatogram of TMS derivatives is given in Fig. 4. Neutral monosac-
charides generally provide several peaks corresponding to pyrano, furano, α, and β forms.
1. Use a FID gas chromatograph and a glass solid injector (moving needle).
2. Use a capillary column (25 m × 0.33 mm) of silicone OV 101.
3. Use carrier gas helium at a pressure of 0.5 bar.
4. Program the oven temperature from 120 to 240°C at 2°C/min.
5. Use injector and detector temperatures of 240 and 250°C, respectively.
168Michalski and Capon
168
Fig. 4. GLC separation of trimethylsilylated deriviatives of monosaccharides released by methanolysis from β-eliminated intes-
tinal mucin oligosaccharide-alditols.
Monosaccharide Composition of Mucin 169
3.2.5. GLC Analysis of Monosaccharides as Alditol Acetates
(26)
(Note 3)
3.2.5.1. H
YDROLYSIS AND
D
ERIVATIZATION
1. Hydrolyze an amount of glycoprotein corresponding to 10 µg of total sugars with 100 µL
of 4 M TFA in the presence of 2 µg of mesoinositol used as internal standard at 100°C for
4 h in glass tubes fitted with a Teflon screw cap (see Note 4).
2. After cooling, evaporate the solution and place the tube in a vacuum dessicator over P
2
O
5
.
3. Reduce the liberated monosaccharides for 1 h at room temperature with 100 µL of a solu-
tion of sodium borohydride (2 mg/mL of 0.05 M ammonia solution).
4. Destroy the excess sodium borohydride by adding of a 20% acetic acid solution until
reaching pH 4.0. Eliminate borate by codistillation with methanol (three times) in a rotary
evaporator.
5. Take up the residue in 0.5 mL of water and freeze-dry.
6. Peracetylate the reduced monosaccharides by adding of 50 µL of pyridine and 50 µL of
acetic anhydride, and leave overnight at room temperature.
7. Remove the excess of reagent under a stream of nitrogen and take up the residue in 50 µL
of dichloromethane containing 1% of acetic anhydride.
3.2.5.2. G
AS
C
HROMATOGRAPHY
C
ONDITIONS
A typical chromatogram is presented in Fig. 5.
1. Use a gas chromatograph equipped with FID detector.
2. Use a glass capillary column (12 m × 0.22 mm) of silicone BPX70.
3. Use: helium at a pressure of 0.6 bar as the carrier gas.
4. Program the oven temperature from 150 to 230°C and 3°C/min and then 230 to 250°C at
5°C/min.
5. Use injector and detector temperatures of 240 and 250°C, respectively.
6. Use an injection volume of 1 µL.
3.3. Separation of Monosaccharides by HPLC
3.3.1. Separation of Native Monosaccharides by HPLC Using Amino-
Bonded Silica (Kromasil-NH
2
)
A typical separation diagram is given in Fig. 6.
1. Inject 10 µL of a monosaccharide mixture (1 mg/mL [w/v]) on to a 5-µm Kromasil-NH2
column (250 × 4.6 mm).
2. Elute with acetonitrile:water (75:25 v/v) at a flow rate of 1 mL/min
3.3.2. Separation of Pyridylamino Derivatives of Monosaccharides by
Reverse-Phase HPLC
(27)
(see Note 5)
Figure 7 presents a typical profile.
1. Hydrolyze oligosaccharides as previously described (see Subheading 3.2.).
2. Evaporate off the solvent and dissolve the residue in 100 µL of coupling reagent (pre-
pared by dissolving 100 mg of 2-aminopyridine in 50 µL of acetic acid and 60 µL of
methanol).
3. Heat the reaction mixture at 90°C for 30 min.
4. Evaporate the reaction mixture under nitrogen with the addition of toluene to remove
excess reagent.
5. Dissolve the pyridylamino monosaccharides in water.
170 Michalski and Capon
Fig. 5. Quantitative determination of monosaccharides by GLC in the itol-acetates form.
6. Apply samples to an analytical Ultrasphere ODS (C18) 5-µm HPLC column (4.6 × 250
mm) (Zorbax).
7. Elute with 0.25 M sodium citrate buffer, pH 4.0, containing 1% acetonitrile at a flow rate
of 1.5 mL/min.
8. Detect by fluorescence with an excitation wavelength of 320 nm and an emission wave-
length of 400 nm.
3.3.3. HPAEC Analysis of Monosaccharides (Notes 6–11)
3.3.3.1. HPAEC S
EPARATION OF
N
EUTRAL
M
ONOSACCHARIDES AND
H
EXOSAMINES
(28)
1. Filter 1 L of Milli-Q water and transfer to bottle 1 as eluent 1. Prepare 50 mM sodium
hydroxide solution by suitable dilution of the 50% sodium hydroxide solution. Connect
the sparge lines and degas for 30 min.
2. Disconnect the sparge lines and pressurize bottles 1 and 2.
3. Run 100% of each solvent through the lines to remove any bubbles.
4. Connect the Carbopac PA-1 column with the guard column.
5. Apply an isocratic run at a flow rate of 1.0 mL/min (see Table 1).
6. Start the system and wait until a stable backpressure is attained.
7. Turn on the PAD detector and wait for stabilization (15 min).
8. Check the baseline and collect chromatographic data by using an integrator.
9. After stabilization and the isocratic run, inject 10 µL of a mixture of standards containing
250 pmol of neutral monosaccharides and hexosamines. Determine the response factors
using the areas.
Monosaccharide Composition of Mucin 171
Fig. 6. Separation of native monosaccharides on an amino-bounded column. 1, Rhamnose;
2, xylose; 3, glucose; 4, mannose; 5, sucrose (internal standard); 6, galactose.
10. Check for good resolution between monosaccharides, their molecular responses, and the
correct retention times (<30 min/run) before injecting the sample.
11. Inject the sample via a Rheodyne valve equipped with a 200- to 500-µL sample loop.
12. Quantify each monosaccharide in the sample by comparison with the injected area of
standards.
3.3.3.2. HPAEC S
EPARATION OF
N
EUTRAL
M
ONOSACCHARIDES
, H
EXOSAMINES
,
AND
U
RONIC
A
CIDS
(28)
1. Add a postcolumn reservoir and deliver NaOH (300 mM) via a mixing tee at a flow rate of
1 mL/min.
2. Follow the steps described in Subheading 3.3.3.1.
3. After each run, equilibrate with the starting eluent in order to yield highly reproducible
retention times for the monosaccharides.
172 Michalski and Capon
4. Inject 10 µL of the mixture of standards containing 250 pmol each of the neutral monosac-
charides, hexosamines, and the required uronic acids.
5. Apply the gradient in Table 2 with eluents 1–4 at a flow rate of 1 mL/min.
6. Quantify the monosaccharides contained in the sample.
3.3.3.3. HPAEC S
EPARATION OF
S
IALIC
A
CIDS
(7)
1. Connect a postcolumn solution of 300 mM sodium hydroxide at a flow rate of 1 mL/min
2. Follow steps 1–8 as described in Subheading 3.3.3.1.
3. Inject 10 µL of a mixture containing 50 pmol of each sialic acid and check the resolution.
The total running time is below 30 min.
4. Load the sample and start the run.
5. Apply a two-step run at a flow rate of 1.0 mL/min (see Table 3).
Fig. 7. HPLC separation of pyridylamino derivatives of monosaccharides by reversed-phase
HPLC Ultrasphere ODS (4.6 × 250 mm). 1, PA-Gal; 2, PA-Glc; 3, PA-Man; 4, PA-Rib; 5, PA-
Fuc; 6, PA-Rham; 7, PA-ManNAc; 8, PA-deoxy-Rib; 9, PA-GlcNAz; 10, PA-GalNAc; 11, PA-
NeuAc.
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