Peptide n glycosidase a

Cellular or larval homogenates were proteolysed either with thermolysin in the case of the larvae or pepsin in the case of High Five cells (20 (link)–22 (link)), prior to cation exchange and gel filtration chromatography of the proteolysate. Thereafter, N-glycans were released from glycopeptides using peptide:N-glycosidase F (PNGase F; Roche) as previously described (21 (link),22 (link)), with a subsequent digestion of the remaining glycopeptides using peptide:N-glycosidase A (PNGase A; Roche). After an initial purification by cation-exchange chromatography (Dowex AG50; flow-through), the glycans were subject to solid-phase extraction on non-porous graphitised carbon (SupelClean ENVICarb, Sigma-Aldrich) as described (22 (link),23 (link)); the ‘neutral’ and ‘anionic-enriched’ fractions were subsequently eluted with 40% acetonitrile and 40% acetonitrile containing 0.1% trifluoroacetic acid respectively. The pools of glycans were then subjected to reductive amination using 2-aminopyridine (PA) (21 (link)). Refer to the Supplement for a Scheme depicting the workflow as well as for further explanations regarding the glycomic analyses and assignments (see also Ref. 22 (link)).

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.