Glycoproteins or blood serum samples were first mixed with PBS buffer (20mM, pH 7.5) and thermally denatured in a 90 °C water bath for 20 minutes. After the denatured sample was cooled to ambient temperature, about 100 units of PNGase F was added to each sample and 2007incubation was conducted in a 37 °C water bath for 18 hours (73 (link)). After PNGase F digestion, protein was removed by precipitation using 90% ethanol. The purified glycans were then subjected to reduction using a procedure based on a method for the reductive β-elimination of O-linked oligosaccharides (74 (link)). An aqueous 10 µg/µL ammonium borane complex solution was prepared. Ten µL of ammonium borane solution was added to each sample and incubated in a 60 °C water bath for one hour. After incubation, 400 µL of methanol was added to each sample which was then dried using a centrifugal vacuum concentrator. This step was repeated 3 to 4 times until excess ammonium borane was removed. The dried reduced sample was resuspended in 1.2 µL of water, 30 µL DMSO and 20 µL of iodomethane for permethylation. A solid-phase permethylation protocol was utilized in this study (57 (link)). A spin column was pack by transferring DMSO soaked sodium hydroxide beads using a micropipette; then DMSO was forced out by centrifugation. The sample mixture was then applied to the prepared reaction spin column. After 25 minutes of incubation, 20 µL of iodomethane was added to the spin column. After another 15 minutes of incubation, the permethylated glycans were eluted using 100 µL of acetonitrile. The eluent was then dried using a centrifugal vacuum concentrator because of the existence of DMSO and a high concentration of salts. Finally, the dried sample was resuspended in a 20% acetonitrile, 0.1% formic acid solution for LC-MS/MS analysis.