Blotglyco h beads

Ammonium bicarbonate 99% (ABC), 1-propanesulfonic acid, 2-hydroxyl-3-myristamido (PHM), 3-Methyl-1-p-tolyltriazene (MTT), and disialyloctasaccharide were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Peptide N-glycosidase F (PNGase F) was purchased from New England Biolabs^R Inc. (Ipswich, Massachusetts, USA), whereas BlotGlyco H beads were purchased from Sumitomo Bakelite, Co. Ltd. (Tokyo, Japan). Dithiothreitol (DTT), iodoacetamide (IAA), O-benzylhydroxylamine hydrochloride (BOA), α-cyano-4-hydroxycin-namic acid (CHCA) and trypsin were from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Other reagents and solvents were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), unless otherwise stated. Protein G spin plate was purchased from Thermo Scientific. SweetBlot™ (automated glycan processing and incubating machine) was from System Instruments Co., Ltd. (Hachioji, Japan). Multi Screen Slvinert^R filter plates were purchased from Millipore Co., Inc. (Tokyo, Japan). All mass quantifications were done using MALDI-TOF/MS (Ultraflex III, Brukers Daltonics, Germany).

N-glycans were analyzed as described13 (link)14 (link). In short, a 250-μL aliquot of BlotGlyco H beads (Sumitomo Bakelite Co., Tokyo, Japan) was reacted with 20 μL PNGase F-digested sample, adjusted to 100 μg protein/20 μL water, with 180 μL of 2% acetic acid in acetonitrile (ACN) at 80 °C for 45 min so that the total glycans were specifically captured by the beads via stable hydrazone bonds. The beads were serially washed and unreacted hydrazide functional groups on the beads were capped by incubation with 10% acetic anhydride in MeOH for 30 min at room temperature. After serial washing, on-bead methyl esterification of the carboxyl groups of sialic acids was carried out by incubation with 150 mM 3-methyl-1-ptolyltriazene in dioxane at 60 °C until dryness. The beads were then serially washed and subjected to transiminization by incubation with aminooxy-functionalized tryptophanyl arginine (ao-WR) for 45 min at 80 °C. The ao-WR-tagged glycans were released by adding 100 μL of water, and then purified using a Mass PREP™ HILIC μElution Plate (Waters, Milford, MA) according to manufacturer instructions.