Peptide n glycosidase f

Total cellular N-glycans were released as described13 (link)24 (link) with minor modifications. Trypsinized cells were lysed by 10% of Triton X-100 for 50 min on ice and sonicated at room temperature for 10 min. The lysates were centrifuged and the supernatants incubated with cold acetone (final concentration 80%) overnight at –20 °C to precipitate the protein fraction. After washing, the cell precipitates were dissolved with 0.02% of 1-propanesulfonic acid and 2-hydroxyl-3-myristamido (PHM) and incubated at 60 °C for 10 min. Aliquots (20 μL) of the cell culture supernatants were also mixed with the same PHM-containing buffer. The samples were reduced by incubation in 10 mM 1,4-dithiothreitol (DTT) at 60 °C for 30 min, and then alkylated by incubation with 20 mM iodoacetamide in the dark at room temperature for 30 min. The mixture was incubated with 80 U trypsin at 37 °C overnight, followed by heat-inactivation. After cooling to room temperature, the N-glycans were released from the trypsin-digested glycopeptides by overnight incubation at 37 °C with 2 U Peptide-N-glycosidase F (PNGase F, Roche). Samples were then dried in a SpeedVac and stored at –20 °C until use. Protein concentrations were determined for all lysates and supernatants by BCA protein assay kit (Thermo Fisher Scientific) according to manufacturer instructions.

10⁷ cells per each cell line were collected. Allantoic and amniotic membranes were extracted from 10-day old chicken embryos. All samples were treated as described previously^{37 (link),59 (link)}. Briefly, each cell line was subjected to sonication in the presence of detergent 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, #10810118001, Roche), reduction in 4 M guanidine-HCl (#24115, Thermo), carboxymethylation, and trypsin (#T0303, Sigma) digestion. The digested glycoproteins were then purified by plus short HLB-Sep-Pak (#186000132, Waters Corp.). N-glycans were released by peptide N-glycosidase F (E.C. 3.5.1.52; #11365177001, Roche) digestion. Released N-glycans were permethylated using the sodium hydroxide procedure and purified by classic short C₁₈-Sep-Pak (#WAT051910, Waters). Permethylated N-glycans were eluted at the 50% acetonitrile fraction.

Sodium dodecyl sulphate (SDS), ethanol and trifluoroacetic acid were purchased from Merck (Darmstadt, Germany). Nonidet P-40 substitute (NP-40), super-DHB (9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid)43 , sodium hydroxide (NaOH) and 1-hydroxybenzotriazole hydrate (HOBt) were acquired from Sigma-Aldrich (Steinheim, Germany). 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was acquired from Fluorochem (Hadfield United Kingdom). Peptide-N-glycosidase F (PNGase F) was supplied by Roche Diagnostics (Mannheim, Germany). Cotton thread was bought from Pipoos (Utrecht, The Netherlands). HPLC SupraGradient acetonitrile (ACN) was acquired from Biosolve (Valkenswaard, The Netherlands). Peptide calibration standard and an AnchorChip MALDI target plate were purchased from Bruker Daltonics (Bremen, Germany). Pooled plasma from 20 healthy human donors was purchased from Affinity Biologicals (Ancaster, Canada) and used as control sample. Lastly, water was purified with a Purelab Ultra from Elga LabWater (Ede, The Netherlands).

All samples were subjected to glycomic profiling as described in a previous study by Biskup et al. without any modification [11 (link)]. Briefly, N-glycans were released and isolated from 10 μl of sample, e.g., the serum/ plasma was diluted in phosphate buffer (pH 6.8), reduced with dithioerythritol (DTE; Sigma-Aldrich) and alkylated with iodoacetamide (IAA; Sigma-Aldrich). Samples were prepared in triplicate. Excess of DTE was used to stop the reaction and 100 mU of Peptide-N-glycosidase F (PNGase F; EC 3.5.1.52; Roche Applied Science, Indianapolis, IN) were used for N-glycan release at 37°C overnight. Samples were purified using C18 cartridges and desalting was performed by graphitized carbon columns (both purchased from Alltech, Deerfield, IL).