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N-glycan release and labeling

HC Hong-Yeol Choi
HP Heajin Park
JH Jong Kwang Hong
SK Sun-Dal Kim
JK Jun-Young Kwon
SY SeungKwan You
JD Jonghye Do
DL Dong-Yup Lee
HK Ha Hyung Kim
DK Dong-Il Kim
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Enzymatic deglycosylation was performed according to a slightly modified method of Hwang et al.22 rrhGAA samples were treated with trypsin (Sigma-Aldrich) and chymotrypsin (Sigma-Aldrich) in 10 mM Tris–HCl buffer, pH 8.0, at 37 °C for 18 h. N-glycans were liberated by glycoamidase A (Roche Diagnostics, Mannheim, Germany) in citrate-phosphate buffer, pH 5.0, at 37 °C for 18 h and purified using a graphitized carbon cartridge (Alltech, Deerfield, IL, USA)23. The released N-glycans were fluorescently derivatized via reductive amination with 2AB (Sigma-Aldrich) according to the method described by Hwang et al. method22. The excess labeling reagent was removed by solid phase extraction packed with a microcrystalline cellulose (Sigma-Aldrich). The purified glycans were lyophilized and stored at −20 °C.

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