Milk oligosaccharides ~ Preparation of oligosaccharides from human milk

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

 Take each 5 mL supernatant into a tube.

 Add 10 mL of methanol and 15 mL of chloroform.

 Shake well and leave at 4°C.

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

 Collect upper layer.

 Evaporate solvent and dissolve with water (a crude oligosaccharide fraction).

 Apply the crude carbohydrate fraction to a Sephadex G-25 column (Medium grade, 5.0 cm ID × 67 cm).

 Elute with water and collect in 20 mL-fractions.

 Take aliquots and dilute with water to 100 μL.

 Add 100 μL of 5% aqueous phenol and 0.5 mL of H₂SO_4.

 Keep for 20 min at room temperature.

 Measure OD490nm (detect hexoses).

 Pool the fractions contain oligosaccharides larger than lactose.

 Apply to a Super Q-Toyopearl 650M column (1.6 × 10 cm).

 Elute with water and collect as a neutral oligosaccharide fraction.

 Elute with 100, 200, 300, 400, 500 mM pyridinium acetate buffer (pH 5.0) with stepwise manner and collect as acidic oligosaccharide fractions.

 Apply a neutral oligosaccharide fraction to a Sephadex G-25 column (Medium grade, 5.0 cm ID × 67 cm).

 Elute with water and collect fractions.

 Take aliquots and spot on a MALDI plate.

 Add 2,5-Dihydroxybenzoic acid (10 mg/mL in 40% (v/v) acetonitrile-water) as a matrix.

 Dry mixture.

 Measure by using MALDI-TOF MS.

 Pool fractions depending on molecular sizes.

 Labeling of oligosaccharides with a sensitive fluorescent derivative is useful for further separation and structural analysis because above each fraction contains several oligosaccharides.

 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 80ºC for 20 min.

 Neutralize by addition of dilute NaOH solution.

 Add 30 μL of 1.7 M NaBH₄ solution.

 Incubated at 40ºC for 20 min.

 Neutralize by addition of dilute acetic acid solution.

 Add water and an equal volume of chloroform.

 Shake well.

 Centrifuge for 5 min.

 Discard lower chloroform layer.

 Add new chloroform and repeat extraction procedure.

 Take upper aqueous phase including the PBH-labeled oligosaccharide.

 Dry by using a vacuum centrifuge.

 Dissolve in water.

 Applied onto a Sep-Pak light C18 cartridge.

 Wash with five bed-volume of water.

 Elute with five bed-volumes of water : acetonitrile = 6:4 (v/v) as PBH-labeled oligosaccharides.

 Evaporate solvent and store at −30ºC in the dark until use.