Pngase f

The purified α-GalNAcase I and II (10 μg) were denatured in 50 μl sodium phosphate (50 mM) buffer, pH 7.0 by heating at 100 °C for 10 min in presence of 0.02% SDS and 10 mM β-mercaptoethanol. The denatured protein was treated with Triton X-100 (Conc. 1.5%) and incubated at 37 °C with 1 unit of PNGase F (Sigma) for 24 h. The PNGase F treated enzyme were separated on SDS-PAGE and stained with CBB R-250 solution.

The rehydrated gel pieces containing purified TBEV E protein were incubated with PNGase F (3 mU, Asparia Glycomics) at 37 °C for 3 h, followed by sonication (5 min) to enrich the supernatant with released oligosaccharides.
Uninfected UKF-NB4 and IRE/CTVM19 cells were first washed with 70% ethanol (100 μL) and after 5 min of sonication, the ethanol solution was evaporated under vacuum. Then the samples underwent extraction as described previously^{28 (link)}. Briefly, chloroform–methanol-water solution in ratio 8:4:1 (~ 300 µL) was added to each sample and sonicated for 5 min. After centrifugation (5 min at 3,000 × g), supernatants were discarded and traces of organic solvent were removed from pellets by evaporation under vacuum. Dried pellets were resuspended in 5 mM AB and incubated with PNGase F for 3 h.
Half of the each PNGase F digested mixture was further incubated with neuraminidase (Clostridium perfringens, Sigma) at 37 °C for 2 h to remove sialic acids. Prior to purification, all digests were analyzed for the presence of N-glycans by applying on-target labeling with phenylhydrazine (PHN) according to the protocol described in the section on MS.

Each C-type lectin (50 μg mDectin-1, mDectin-2, and SIGN-R1) was incubated in 50 mM ammonium bicarbonate buffer with 10 U PNGase F (recombinant PNGase F of Flavobacterium meningosepticum; MilliporeSigma) for 24 h at 37°C. The reaction was stopped by freeze drying. The released N-glycans were then purified on C18-SepPak cartridges (Waters). Cartridges were activated by 5 ml methanol, equilibrated with 5 ml acetic acid (5%, v/v), 5 ml acetonitrile, and 10 ml of acetic acid (5%, v/v). Samples were solubilized into 200 μl acetic acid (5%, v/v) and loaded onto the cartridges. The flow-through was collected, and cartridges were washed with 10 ml acetic acid (5%, v/v) and the solutions freeze dried before the permethylation of purified N-glycans.

Twenty μg of the N-terminal peptide (aa 1–220), expressed in HEK293 cells, were deglycosylated in 80 μl of PBS pH7.4 and 1.6 μl of neuraminidase (Cat.# 1158886001; Millipore-Sigma) and 1.6 μl PNGaseF (Cat.# 9109-GH; R&D Systems), incubated for 1 hour at 37°C and then at 4°C overnight.

To assess the glycosylation pattern of the UUKV glycoproteins, virus stocks purified through a 25% sucrose cushion were denatured and exposed to one of the five following treatments: 1,000 units of endoglycosidase H (Endo H; Promega), 5 units of peptide-N-glycosidase F (PNGase F), 0.005 units of α-2(3, 6, 8, 9)-neuraminidase, 0.003 units of β-1,4-galactosidase, and 0.05 units of β-N-acetylglucosaminidase (all enzymes from Merck Millipore) according to the manufacturer's recommendations. Samples were then analyzed by SDS-PAGE on a 4 to 12% or 10% Bis-Tris NuPAGE Novex gel (Life Technologies) and immunoblotting.

For one aliquot of the enriched N-linked intact glycopeptides (4 μg) from unfractionated serum proteins, 1 μl of PNGase F (P0705S, 500,000 units/ml, NEB, MA) was added into 10 μl of N-linked intact glycopeptide solution in 50 mm NH₄HCO₃ and incubated 4 h at 37 °C to release N-glycan. After PNGase F treatment, the deglycopeptide samples were desalted by ZipTip C18 (ZTC18S960, Merck KGaA, Darmstadt, Germany) with 0.1%TFA and eluted successively with 10 μl 20% ACN/0.1%TFA, 10 μl 40% ACN/0.1%TFA, 10 μl 60% ACN/0.1%TFA. The eluted fractions were combined and dried by refrigerated centrifugal vacuum concentrator and re-dissolved in 20 μl 0.1% FA for LC-MS/MS analysis.
For one aliquot of enriched N-linked intact glycopeptides (40 μg) from fractionated sera proteins, 10 μl of PNGase F was added into 40 μl of N-linked intact glycopeptide solution in 50 mm NH₄HCO₃ and incubated 4 h at 37 °C to release N-glycan. After PNGase F treatment, the deglycopeptide samples were desalted by C18 Tips (87784, Pierce, Rockford) with 0.1%TFA and eluted successively with 100 μl 20% ACN/0.1%TFA, 100 μl 40% ACN/0.1%TFA, 100 μl 60% ACN/0.1%TFA. The eluted fractions were combined and dried by refrigerated centrifugal vacuum concentrator and re-dissolved in 20 μl 0.1% FA for LC-MS/MS analysis.