Rapid Labeling and Purification of N-Glycans

Release of N-glycan, Rapiflour labeling and purification of Rapiflour labeled N-glycan were performed according to the manufactures’ protocols. Briefly, 15 µg of each enzyme was heat denatured at 90°C for 3 min in 6 µL of buffer solution containing 5% (w/v) RapiGest SF and 18.2 megohm water. After cooling down to room temperature enzymes were deglycosylated with 1.2 µL of RapiPNGase F at 50°C for 5 min. Thereafter, the enzymes were labeled with 12 µL of the RapiFlour-MS Reagent Solution at room temperature for 5 min and 358 µL of acetonitrile solution was added to dilute the reaction. To enrich the glycans, hydrophilic interaction liquid chromatography solid phase extraction (HILIC SPE) was performed by Waters GlycoWorks HILIC µElution Plate. The plate was washed with 200 µL of Milli-Q water, followed by equilibration with 200 µL of 15:85 water/acetonitrile. After loading the acetonitrile-diluted sample, the well was washed twice with 600 µL of 1:9:90 (v/v/v) formic acid/water/acetonitrile. The glycans were eluted with 30 µL of GlycoWorks SPE Elution Buffer (200 mM ammonium acetate in 5% acetonitrile).