Ascentis express 2.7 μ rp amide column

Separation of pyridylamino-labeled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 10 AXL; excitation at 320 nm and emission 400 nm). For standard RP-HPLC, a Hypersil ODS column (Agilent; a classical C18 column of the dimensions 250 × 4.6 mm) was used with 100 mM ammonium acetate, pH 4.0 (buffer A) and 30% (v/v) methanol (buffer B); a gradient of increasing buffer B (1% per minute) was programmed up to 30 minutes; a step up to 40% B for 5 minutes was followed by another step to 45% for 5 minutes followed by a return to starting conditions. A similar linear gradient (1% B per minute; 0.8 ml/min) for 45 minutes was used with a Kinetex™ 5μ XB-C18 column (250 × 4.6 mm; Phenomenex), which has iso-butyl side chains on fused core particles. For the Ascentis® Express 2.7 μ RP-Amide column (150 × 4.6 mm; Sigma-Aldrich) which has a fused core carrying alkyl amide chains as the bonded phase, a gradient of buffer B 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. The recommended guard columns were used in all cases. The overall HPLC and MALDI-TOF MS results from two independently-grown nematode samples were similar, but only the data for one preparation are shown.

Adult Haemonchus contortus were obtained from infected sheep and the N-glycans were prepared using standard laboratory protocols (Paschinger, et al. 2012 (link)) with release of N-glycans using peptide:N-glycosidase A from peptic peptides. After pyridylamination and two rounds of gel filtration, the labelled N-glycans were fractionated by HPLC using an Ascentis^® Express 2.7 μ RP-Amide column (150 × 4.6 mm; Sigma-Aldrich)(Yan, et al. 2015 ). Monoisotopic MALDI-TOF MS was performed using a Bruker Autoflex Speed (equipped with a 1000 Hz Smartbeam™-II laser) instrument in positive reflectron mode with 6-aza-2-thiothymine (ATT) as matrix. MS/MS was performed by laser-induced dissociation. Further analysis by MALDI-TOF MS was performed after treatment with either Aspergillus β-galactosidase (Dragosits, et al. 2014 (link)), bovine kidney α-fucosidase (Sigma-Aldrich), α-mannosidases (jack bean from Sigma or Xanthomonas α1,2/3-specific from New England Biolabs), α-galactosidase (coffee bean from Sigma-Aldrich) or recombinant Apis mellifera FDL β-hexosaminidase (Dragosits, et al. 2015 ) in 25 mM ammonium acetate, pH 4.5, at 37 °C overnight (three hours in the case of FDL). For removal of α1,3-fucose or phosphorylcholine residues, selected fractions were dried and incubated for 24 or 48 hours at 0 °C with 3 μl 48% (v/v) hydrofluoric acid prior to evaporation.

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.