Trichuris suis adults (kindly supplied by Dr. Stig Thamsborg of the Department of Veterinary Disease Biology, University of Copenhagen, Denmark) were homogenised and proteolysed with pepsin. N-glycans were then released from peptic peptides using peptide:N-glycosidase A (Roche) according to the procedures described previously [28 (link)]. Free glycans were labelled with 2-aminopyridine [29 (link),28 (link)] prior to MALDI-TOF MS and fractionation by reversed-phase HPLC (RP-HPLC). Separation of PA-labeled glycans was performed on a Shimadzu HPLC system equipped with a fluorescence detector (RF 10 AXL; excitation at 320 nm and emission at 400 nm) and an Ascentis® Express 2.7 μ RP-Amide column (150 × 4.6 mm; Sigma-Aldrich). A gradient of 30% methanol (solvent B), with 100 mM ammonium acetate, pH 4.0, as buffer (solvent A), up to 35% over 34 minutes was applied at a flow rate of 0.8 ml/min as follows: 0-4 min, 0% B; 4-14 min, 0-5% B; 14-24 min, 5-15% B; 24-34 min, 15-35% B; 34-35 min, return to starting conditions [27 (link)]. The RP-HPLC analysis of the glycan preparation was performed twice together with MALDI-TOF MS of each fraction.