Glycobuffer 1

Pre-formed fixed Detroit 562 pharyngeal cell monolayers of the 96-well microtiter plate were treated with 30 µL/well reaction volumes of each of the exoglycosidases. For mannosidases, the reaction volume comprises 3 µL 1 × GlycoBuffer 1 (NEB, Notting Hill, Australia), 0.3 µL 100 µg/mL purified BSA (NEB, Notting Hill, Australia), 0.2 µL α1-6 mannosidase (8 U) or α1-2,3 mannosidase (8 U) (NEB, Notting Hill, Australia), and 26.5 µL PBS. For Sialidase A, 6 µL 5 × Reaction Buffer B (250 mM sodium phosphate pH 6.0) (Prozyme, Hayward, CA, USA), 0.2 µL Sialidase A (1 × 10⁻³ U) (Prozyme, Hayward, CA, USA), and 23.8 µL PBS. Untreated PBS wells representing the intact surface glycome of the fixed Detroit 562 pharyngeal cell monolayers were also included as a control. The plate was incubated for 2 h, 37 °C. Once incubated, the wells were washed once with PBS. Glycan removal was confirmed via lectin binding assay as per [25 (link)], with exoglycosidase pre-treated pharyngeal cell monolayers incubated with either biotinylated Hippeastrum hybrid lectin (binding mannose residues) (Vector Laboratories, Burlingame, CA, USA) or biotinylated Sambucus nigra lectin (binding sialic acid residues) (Vector Laboratories, Burlingame, CA, USA) (Supplementary Material 5, Figure S2).

N-acetyl-β-d-glucosaminidase S from Streptococcus pneumoniae (New England Biolabs, Ipswich, MA, USA) and α1,3/4-l-fucosidase from Streptomyces sp. 142 (Takara Bio, Shiga, Japan) were used in 1 × GlycoBuffer 1 (50 mM sodium acetate and 5 mM CaCl₂ pH 5.5; New England Biolabs) at 37 °C overnight. β4-d-Galactosidase from S. pneumoniae (Agilent, Santa Clara, CA, USA) was incubated in sodium citrate buffer (pH 6.0) at 37 °C. Sodium acetate (100 mM pH 5.0) with 2 mM zinc chloride was used as the buffer for α1, 2/3/6-mannosidase from Jack Bean (Agilent). Sodium phosphate (100 mM pH 6.8) was used as the buffer for α-xylosidase from E. coli (Megazyme, Bray, Ireland). Purified N-glycans released from α1-acid glycoprotein in human plasma (Sigma-Aldrich, St Louis, Mo, USA) with Endo F3 (New England Biolabs) were labeled with 2-aminopyridine for use as standard glycans. The standard for fucosylated N-glycan with an α1,3-mannose arm extended β4-branching lactosamine was prepared by enzymatic digestion of triantennary N-glycan with sialidase, galactosidase, hexosaminidase and mannosidase, without fucosidase treatment.