Each sample (100 μg) was added to 100 μL digestion buffer (50 mmol/L pH 7.0). Heparin
lyase II (10 mU in Tris–HCl buffer, pH 7.0) was added and the sample was digested in 37°C
water bath for 12 hours to produce fragments. Enzymatic digestion was terminated by
removing the enzymes using an YM-3 centrifugal filter unit. The filtrates were lyophilized
and redissolved in 100 µL of distilled water at a concentration of 1 µg/mL. Online
hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS)
was used to analyze the resulting oligosaccharides.12 (link) A Luna HILIC column (2.0 mm2 × 50 mm2, 200 Å; Phenomenex,
Torrance, California) was connected online to the standard ESI source of LTQ-Orbitrap XL
FTMS (Thermo Fisher Scientific, San Jose, California). Mass spectra were acquired at a
resolution 60 000 with 200 to 1800 m/z range.
lyase II (10 mU in Tris–HCl buffer, pH 7.0) was added and the sample was digested in 37°C
water bath for 12 hours to produce fragments. Enzymatic digestion was terminated by
removing the enzymes using an YM-3 centrifugal filter unit. The filtrates were lyophilized
and redissolved in 100 µL of distilled water at a concentration of 1 µg/mL. Online
hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS)
was used to analyze the resulting oligosaccharides.12 (link) A Luna HILIC column (2.0 mm2 × 50 mm2, 200 Å; Phenomenex,
Torrance, California) was connected online to the standard ESI source of LTQ-Orbitrap XL
FTMS (Thermo Fisher Scientific, San Jose, California). Mass spectra were acquired at a
resolution 60 000 with 200 to 1800 m/z range.
