Preparation of oligosaccharides from human milk

Materials

1. Column for size-exclusion chromatography filled with gel, such as Sephadex G-25 (medium grade)

2. Anion-exchange column filled with gel, such as TOYOPEARL Super Q-650M (Tosoh Bioscience)

3. 5% (w/v) aqueous phenol

4. H₂SO₄

5. 100, 200, 300, 400, and 500 mM pyridinium acetate buffer (pH 5.0)

6. 10 mg/mL 2,5-dihydroxybenzoic acid in 40% (v/v) acetonitrile-water if using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)

7. Millipore ultrapure water or redistilled water is recommended as water

Instruments

1. Photometer for measuring visible light and plastic disposable cuvette, if possible.

2. Fraction collector

3. Cooling centrifuge with swing bucket

4. Evaporator or Speed Vac

5. MALDI-TOF MS

Methods

For a better understanding, Figure 1 shows the entire process described below and the yield obtained at each step.

1.

Removal of fats and proteins

a.

Centrifuge milk (10–20 mL per tube) for 15 min at 5,000 ×g at 4°C.

b.

Remove the creamy fat layer with a spatula, and transfer the water layer (each ~5 mL) to a new tube.

c.

Add 10 mL of methanol and 15 mL of chloroform.

d.

Shake well and leave at 4°C.

e.

Centrifuge for 5 min at 5,000 ×g at 4°C.

f.

Collect upper layer from all tubes.

g.

Evaporate the solvent to dry and dissolve with 20 mL of water (the crude oligosaccharide fraction)

2.

Gel filtration

a.

Apply the crude oligosaccharide fraction to a Sephadex G-25 column (5 cm of ID × 67 cm) equilibrated with water.

b.

Elute with water under hydrostatic pressure and collect in 20 mL fractions.

c.

Take aliquots and dilute with water to 100 μL.

d.

Add 100 μL of 5% aqueous phenol and 0.5 mL of H₂SO₄.

e.

Keep for 20 min at room temperature.

f.

Measure OD490 nm (detection of hexoses)

g.

Draw a chromatogram plotting the absorbance against the fraction number on the horizontal axis, and find the elution of lactose with the highest peak near the column volume.

h.

Pool the fractions containing oligosaccharides larger than lactose.

3.

Separation of neutral and acidic oligosaccharides

a.

Apply a part of the pooled fraction to a TOYOPEARL Super Q-650M column (1.6 cm of ID × 10 cm) equilibrated with water.

b.

Elute with 40 mL of water under hydrostatic pressure, and collect this pass-through fraction (that is unbound to the column) as a neutral oligosaccharide fraction, N.

c.

Elute with 40 mL of 100, 200, 300, 400 and 500 mM pyridinium acetate buffer (pH 5.0) stepwise under hydrostatic pressure, store the five fractions, and name as acidic oligosaccharide fractions, A1, A2, A3, A4, and A5, respectively (Note 2).

d.

Perform Steps 2c–f to determine the amounts of hexose in the fractions, if necessary.

4.

Size fractionation by gel filtration

a.

Apply the neutral oligosaccharide fraction, N to a Sephadex G-25 column (5 cm of ID × 67 cm)

b.

Elute with water under hydrostatic pressure and collect in 20 mL fractions.

Perform Steps 2c–f to determine the amounts of hexose in the fractions, if necessary.

c.

Take an aliquot of each fraction to detect oligosaccharides and put on a MALDI plate.

d.

Add 2,5-dihydroxybenzoic acid solution as a matrix to the sample and mix and dry the mixture.

e.

Measure using MALDI-TOF MS

f.

Pool fractions depending on molecular sizes, for example, N-1, N-2 (mainly contains oligosaccharides with decaose cores), N-3 (mainly contains oligosaccharides with octaose cores), N-4 (mainly contains oligosaccharides with hexose cores), N-5 (mainly contains oligosaccharides with tetraose cores), and N-6 (mainly contains lactose) (Note 3).

5.

Labeling with pyrene butanoic acid hydrazide (PBH) for further detailed analysis of the structures (Figure 2, Note 4)

a.

Heat oligosaccharides (approximately 1 nmol) and PBH (100 nmol) in 20 μL of methanol in the presence of 0.1 N acetic acid in a glass vial tightly sealed with a screw cap at 80°C for 20 min.

b.

Neutralize by adding dilute NaOH solution.

c.

Add 30 μL of 1.7 M NaBH₄ solution and incubated at 40°C for 20 min.

d.

Neutralize by adding dilute acetic acid solution.

e.

Add 400 μL of water and 500 μL of chloroform.

f.

Shake well and centrifuge for 5 min.

g.

Discard lower chloroform layer.

h.

Add 500 μL of new chloroform and repeat extraction procedure.

i.

Take upper aqueous phase including the PBH-labeled oligosaccharides.

j.

Dry using a vacuum centrifuge.

k.

Dissolve in water and apply onto a Sep-Pak C18 cartridge.

l.

Wash with five-bed volume of water

m.

Elute with five-bed volume of water-acetonitrile (6:4, v/v)

n.

Evaporate solvent and store at −30°C in the dark until use (Note 5).

Notes

1. This protocol is for relatively large-scale preparation of intact oligosaccharides from human milk (use up to 250 mL). An example of the preparation results is shown in Figure 1. When using a bigger quantity, such as one liter, the method by Kobata (11) is recommended.

2. Most monosialylated oligosaccharides are recovered in A1 fraction, and some monosulfated oligosaccharides are found in A2 although detailed structural study is not done.

3. Each fraction by gel filtration contains several oligosaccharides. Separation and detection of closely related or isomeric structures is difficult with using intact oligosaccharides. It is necessary to derivatize oligosaccharides and use a method with better resolution. Lectin column chromatography will be valuable for further separation. Aleuria aurantia lectin-immobilized column chromatography is effective for fucosylated oligosaccharides.

4. Labeling of oligosaccharides with a sensitive fluorescent derivative allows for further separation and structural analysis. There are many labeling reagents, such as 2-aminopyridine and 2-aminobenzamide. Among them, PBH-labeled oligosaccharides (Figure 2) have strong hydrophobicity, and therefore, it can separate isomers on high-performance liquid chromatography (HPLC). The detection sensitivity of PBH-oligosaccharides on MALDI-MS is 1000 times that of intact oligosaccharides. Since many negative ions by MALDI are also generated and can be applied to MS², MS³, and MS⁴, isomers even in the mixture can be identified using diagnostic fragment ions, which are produced from particular structures. The author determined many kinds of fucosylated isomers containing Type 1H, Le^a, and Le^x using this method (2).

5. For example, PBH-labeled oligosaccharides can be analyzed on HPLC with Inertsil WP300 C₁₈ column (GL Sciences) (4.6 × 500 mm) using water-acetonitrile eluent system for neutral oligosaccharides and 50 mM acetic acid-triethylamine (pH 5.0)-acetonitrile eluent system for acidic oligosaccharides. PBH-oligosaccharides are detected at Ex 341 nm/Em 376 nm.