Each 10-μL serum sample aliquot was dissolved in 50 μL of a 106-mM solution of ammonium bicarbonate containing 12 mM 1,4-dithiothreitol and 0.06% 1-propanesulfonic acid, 2-hydroxyl-3-myristamido (Wako Pure Chemical Industries, Osaka,

Japan). After incubation at 60°C for 30 minutes, 123 mM iodoacetamide (10 μL) was added to the mixtures followed by incubation in the dark at room temperature to enable reductive alkylation. After 60 minutes, the mixture was treated with 200 U of trypsin (Sigma-Aldrich, St. Louis, MO) at 37°C for 2 hours, followed by heat-inactivation of the enzyme at 90°C for 10 minutes. After cooling to room temperature, the N-glycans were released from the tryptic glycopeptides by incubation with 325 U of PNGase F (New England BioLabs, JQ1 Ipswich, MA) at 37°C for 6 hours. Glycoblotting of sample mixtures containing whole serum N-glycans was performed in accordance with previously described procedures. Commercially available BlotGlyco H beads (500 μL) (10 mg/ml suspension; Sumitomo Bakelite) were aliquoted into the wells of a MultiScreen Solvinert hydrophilic PTFE (polytetrafluoroethlene) 96-well KU-60019 price filter plate (EMD Millipore, Billerica, MA). After removal of the water using a vacuum pump, 20 μL of PNGase F-digested samples were applied to the wells, followed

by the addition of 180 μL of 2% acetic acid in acetonitrile. The filter plate was then incubated at 80°C for 45 minutes to capture the N-glycans onto the beads by way of a chemically stable and reversible hydrazone bond. The beads were then washed using 200 μL of 2 M guanidine-HCl in 10 mM ammonium bicarbonate, followed by washing with the same volume of water and of 1% triethyl amine in methanol. Each washing step was performed twice. The N-glycan linked beads were next incubated with 10% acetic anhydride in 1% triethyl amine in methanol for 30 minutes

at room temperature so that unreacted hydrazide groups would become capped by acetylation. After capping, the reaction solution was removed under a vacuum and the beads were serially washed with 2 × 200 μL aminophylline of 10 mM HCl, 1% triethyl amine in methanol, and dioxane. This is a pretreatment for sialic acid modification. On-bead methyl esterification of carboxyl groups in the sialic acids was carried out with 100 μL of 100 mM 3-methyl-1-P-tolyltriazene (Tokyo Chemical Industry, Tokyo, Japan) in dioxane at 60°C for 90 minutes to dryness. After methyl esterification of the more stable glycans, the beads were serially washed in 200 μL of dioxane, water, 1% triethyl amine in methanol, and water. The captured glycans were then subjected to a trans-iminization reaction with BOA (O-benzylhydroxylamine) (Tokyo Chemical Industry) reagent for 45 minutes at 80°C. After this reaction, 150 μL of water was added to each well, followed by the recovery of derivatized glycans under a vacuum.