β1 3 galactosidase

All lectins listed were purchased from Vector Laboratories (Burlingame, CA). BSA was purchased from Thermo Fisher Scientific (Waltham, MA). Bovine fetuin, asialofetuin, and native human alpha-1-acid glycoprotein were purchased from Sigma-Aldrich (St. Louis, MO). Native human haptoglobin was purchased from Bio-Rad (Hercules, CA). Anti-Bovine fetuin polyclonal antibody was purchased from GeneTex, Inc. (Irvine, CA). Papain coated magnetic particles were from Spherotech (Lake Forest, IL). Sialidase A 66 was purchased from Agilent (Santa Clara, CA) while β1-3 Galactosidase and β1-4 Galactosidase S were purchased from New England Biolabs (Ipswitch, NH). Additional β1-4 Galactosidase used on human haptoglobin and alpha-1-acid glycoprotein was purchased from Agilent. Alpha-2,6-Sialyltransferase, rec. and α-2,3-Sialyltransferase, rec. were purchased from Roche Diagnostics GmbH (Mannheim, Germany).

Human recombinant full-length MR with His-tag, expressed in mouse myeloma cell line NS0, was purchased from R&D systems, anti-human IgG Alexa-Fluor 488-conjugated from Jackson ImmunoResearch, and anti–penta-His Alexa-Fluor 488 conjugate from Qiagen. Biotinylated Concanavalin A, R. communis agglutinin-I, Maackia amurensis lectin-I, Sambucus nigra lectin, fluorescein Concanavalin A, fluorescein Wheat germ agglutinin, and Man-BSA were purchased from Vector Laboratories; neuraminidase and Protein A-agarose beads from Roche; O-glycosidase, PNGase F, α-galactosidase, β1,3-galactosidase, and β1,4-galactosidase from New England Biolabs; sequencing grade trypsin from Promega; Glucitol-polyacrylamide (PAA), Man₃-PAA, and Glucitol-PAA biotin from Lectinity; GlcNAc and GlcNAc-BSA from Carbosynth. All other chemicals were purchased from Sigma-Aldrich if not indicated differently.

For sequential digestion of N-glycans released from SEAP β-N-acetylglucosoaminidase S, β1–3 galactosidase, β1–4 galactosidase S and α2–3,6,8,9 neuraminidase A were used (all enzymes were obtained from New England Biolabs, Ipswich, MA, USA). 2-AB-labeled and dried N-glycans were dissolved in 10 µL of 1x GlycoBuffer 1 (New England Biolabs, Ipswich, MA, USA) and incubated overnight at 37 °C with exoglycosidase(s) of interest. After digestion, 3 µL of the reaction mixture was mixed with 7 µL of acetonitrile and directly applied onto the GlycoSepN normal-phase amide column.