Ammonium bicarbonate (ABC), Dowex cation-exchange resin (50W-X8), trifluoroacetic acid (TFA), hydrochloric acid (HCl), DL-dithiothreitol (DTT), ammonium acetate, sodium chloride (NaCl) and sodium borohydride (NaBH4) were obtained from Sigma-Aldrich (Steinheim, Germany). The 8 M guanidine hydrochloride (GuHCl) was purchased from Thermo Fisher Scientific. Peptide N-glycosidase F (PNGase F) was purchased from Roche Diagnostics (Mannheim, Germany). Glacial acetic acid and potassium hydroxide (KOH) were purchased from Honeywell Fluka. Solid phase extraction (SPE) bulk sorbent Carbograph was obtained from Grace Discovery Sciences (Columbia, MD, USA). HPLC SupraGradient acetonitrile (ACN) was obtained from Biosolve (Valkenswaard, The Netherlands) and other reagents and solvents such as methanol, ethanol and 2-propanol were purchased from Merck (Darmstadt, Germany). MultiScreen® HTS 96-multiwell plates (pore size 0.45 μm) with high protein-binding membranes (hydrophobic Immobilon-P PVDF membrane) and a 96-well PP Microplate were purchased from Millipore (Amsterdam, The Netherlands), conical 96-well Nunc plates from Thermo Scientific (Roskilde, Denmark) and a 96-well PP filter plate from Orochem Technologies (Naperville, IL, USA). Ultrapure water was used for the all preparations and washes, generated from a Q-Gard 2 system (Millipore, Burlington, MA, USA).
Peptide n glycosidase f pngase f
Manufactured by Roche
Sourced in Germany
Peptide N-glycosidase F (PNGase F) is an enzyme used in the laboratory for the removal of N-linked glycans from glycoproteins. It catalyzes the cleavage of the bond between the asparagine residue and the first N-acetylglucosamine residue of the N-glycan chain. This enzymatic activity is useful for the structural analysis and characterization of glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
96 well pp microplate, by Merck Group (2 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Sodium borohydride nabh4, by Merck Group (1 mentions)
8 m guanidine hydrochloride guhcl, by Thermo Fisher Scientific (1 mentions)
Hplc supragradient acetonitrile acn, by Biosolve (1 mentions)
Q gard 2 system, by Merck Group (1 mentions)
Multiscreen hts 96 multiwell plates, by Merck Group (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
2 propanol, by Merck Group (1 mentions)
Simplicity water purification system, by Merck Group (1 mentions)
Alpha 1 acid glycoprotein from human plasma, by Merck Group (1 mentions)
Lc ms grade methanol, by Avantor (1 mentions)
Absolute ethanol hplc grade, by Avantor (1 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
Sephadex g 25 superfine gel, by GE Healthcare (1 mentions)
Sodium acetate trihydrate, by Avantor (1 mentions)
Sodium cyanoborohydride, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
10 protocols using peptide n glycosidase f pngase f
1
Glycoprotein Characterization Workflow
2
Deglycosylation of C1-Inh Protein
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For samples to be treated with exoglycosidases, de-N-glycosylation was achieved using an in-solution Peptide-N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) approach prior to in-gel digestion with proteolytic enzymes. Ten microliter C1-Inh (1 μg/μl) were first denatured and reduced with a mixture of 1 μl of 5% SDS and 0.4 m DTT by shaking thoroughly for 5 min at 95 °C. Then, 10 μl PNGase F solution (1 U in 2% NP-40/2 x PBS) was added, followed by an overnight incubation at 37 °C. De-N-glycosylated C1-Inh was then subjected to SDS-PAGE and in-gel proteolytic digestion as described below.
3
Recombinant SARS-CoV-2 RBD Glycosylation
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Reagents used for this study were
at least of analytical grade; for more details, seeSupporting Information S1 . Recombinant RBDs (Wuhan-Hu-1-isolate (MN908947)), either transiently expressed
in HEK293 or stably expressed in CHO cells, were used (InVivo Biotech
Services, Henningsdorf, Germany). The constructs contained the amino-acid
sequence 319 to 541 with a C-terminal 6xHis-Tag. Recombinant RBDs
were purified using immobilized metal affinity chromatography and
a size-exclusion polishing step. The samples were stored in 20 mM
sodium phosphate, 300 mM NaCl, pH 7.2. Glycosidases SialEXO (sialidases
α2-3, α2-6, and α2-8), GalactEXO (galactosidases
β1-3 and β1-4), OglyZOR (endo-α-N-acetylgalactosaminidase), OpeRATOR (O-protease), α1-2 fucosidase, and α1-3,4 fucosidase were
obtained from Genovis (Lund, Sweden). Peptide N-glycosidase
F (PNGaseF) was purchased from Roche Diagnostics (Mannheim, Germany).
at least of analytical grade; for more details, see
in HEK293 or stably expressed in CHO cells, were used (InVivo Biotech
Services, Henningsdorf, Germany). The constructs contained the amino-acid
sequence 319 to 541 with a C-terminal 6xHis-Tag. Recombinant RBDs
were purified using immobilized metal affinity chromatography and
a size-exclusion polishing step. The samples were stored in 20 mM
sodium phosphate, 300 mM NaCl, pH 7.2. Glycosidases SialEXO (sialidases
α2-3, α2-6, and α2-8), GalactEXO (galactosidases
β1-3 and β1-4), OglyZOR (endo-α-N-acetylgalactosaminidase), OpeRATOR (O-protease), α1-2 fucosidase, and α1-3,4 fucosidase were
obtained from Genovis (Lund, Sweden). Peptide N-glycosidase
F (PNGaseF) was purchased from Roche Diagnostics (Mannheim, Germany).
4
Glycoprotein Characterization Protocol
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Vacuette® 9 mL tubes without additives were obtained from Greiner Bio-One International GmbH (Kremsmünster, Kirchdorf an der Krems District, Austria). LC-MS grade methanol, acetonitrile (ACN) and formic acid (FA), HPLC grade absolute ethanol, chloroform and ammonia (32%), analytical grade ortho-boric acid, and sodium acetate trihydrate were purchased from VWR Chemicals (Radnor, PA, USA). 1,3-bis[tris(hydroxymethyl)methylamino]propane (bis-tris propane) (98+%) was purchased from Alfa Aesar (Haverhill, MA, USA). Alpha-1-acid glycoprotein from human plasma (≥99%), molecular biology grade K2HPO4, sodium dodecyl sulfate (SDS), ß-mercaptoethanol (ß-ME), and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma Aldrich (St. Louis, MO, USA). Sodium cyanoborohydride (95%) was purchased from Acros Organics (Geel, Belgium). Fractogel TMEA-650 (M) was obtained from Merck (Darmstadt, Germany). AA (≥99%) and molecular biology grade NaCl were obtained from Fisher Scientific (Hampton, NH, USA). Peptide:N-glycosidase F (PNGase F) was purchased from Roche Diagnostics (Mannheim, Germany). Sephadex G-25 Superfine gel was purchased from GE Healthcare (Chicago, IL, USA). Water of Milli-Q (MQ) purity prepared by Simplicity® Water Purification System (Merck Millipore, Burlington, MA, USA) was used.
5
Enzymatic Deglycosylation of Soluble Brain Proteins
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Soluble fraction proteins (30 μg) from mouse brains were precipitated with ethanol and suspended with 20 to 50 μl of chondroitinase buffer (10 mM Tris-HCl (pH7.4), 30 mM sodium acetate, 50 mM EDTA and protease inhibitors), followed by incubation with a final concentration of 250 μU/ml of chondroitinase ABC (chABC) (Seikagaku Corporation) for 3 h at 37°C. The reaction mixtures were denatured with 0.5% sodium dodecyl sulfate (SDS) for 5 min at 100°C. To reduce the SDS concentration, the solution was diluted with 4 volumes of phosphate-buffered saline (PBS) containing 25 mM EDTA, 1.25% NP-40 and 1.25% 2-mercaptoethanol. Peptide N-glycosidase F (PNGase F, Roche Applied Science) was added at the final concentration of 20 mU/μl, followed by incubation for 16 h at 37°C.
6
Deglycosylation and Mass Spectrometry of ECM1
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Recombinant ECM1 purified from conditioned media was denatured using 1% SDS at 95 °C for 5 min and was diluted 1:10 with 1% Triton X-100. To deglycosylate the denatured protein, the sample was treated using peptide N-glycosidase F (PNGaseF; Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer recommendations. Both deglycosylated and untreated samples were subjected to SDS–PAGE. After CBB staining, a visible band was excised and destained. In-gel digestion was performed using trypsin (TPCK-treated, Worthington Biochem. Co., Freehold, NJ). The digestion mixture was separated using a nanoflow LC (Easy nLC, Thermo Fisher Scientific, Waltham, MA) on a PepMap RSLC analytical column (C18, φ50 μm × 15 cm, 2 μm, 100 Å, Thermo Fisher Scientific) with a linear gradient of 0–35% buffer B (100% ACN and 0.1% formic acid) at a flow rate of 300 nL/min over 10 min and subjected on-line to a Q Exactive mass spectrometer (Thermo Fisher Scientific) with a nanospray ion source. MS and MS/MS data were acquired using a data-dependent top5 method. Obtained MS/MS data were searched against an in-house database, including the ECM1 sequence, using the MASCOT program (Matrix Science, London, UK) with variable modifications: Gln → pyro-Glu (N-term Q), Oxidation (M), Propionamide (C), Hex (W).
7
Transferrin Purification and N-Glycan Analysis
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Each transferrin was purified from 120 μL of serum sample by immunoaffinity separation on transferrin depletion columns pre-packed with anti-TfIgY beads (GenWay Biotech, San Diego, CA, USA) as already described in detail [25] . The eluted transferrin solutions were concentrated to 100 μL in 50 mM ammonium bicarbonate buffer, 1 μL of intact transferrin solution was then analyzed by MALDI-MS in linear mode and positive polarity by using 5α-Cyano-4hydroxycinnamic acid (CHCA), 10 mg/mL in 60/40 (v/v) 0.1% TFA/ACN, as matrix (1:20 sample/matrix). No trace of contaminant proteins was detected. The remaining sample was then deglycosylated by peptide-N-glycosidase F (PNGase F, Roche Molecular Biochemicals, Mannheim, Germany), purified and permethylated following the same protocol adopted for total serum N-glycan analysis.
8
Immunoprecipitation and Glycosidase Treatment
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For immunoprecipitation, primary antibodies (20 μg/mL) and protein G-Sepharose 4 Fast Flow (Cat# 17061801, GE Healthcare, Chicago, IL, USA) were added to the soluble fractions and rotated at 4°C for 2 h. Following centrifugation at 3000 × g for 2 min, the supernatant was obtained as an unbound fraction. Precipitated proteins were eluted with 1% SDS, giving rise to the bound fraction. For glycosidase treatment, proteins of the soluble fraction of the mouse brain were precipitated with ethanol, re-suspended, and denatured with 0.5% SDS at 100°C for 5 min. Then, to reduce the concentration of SDS, the solution was diluted with four volumes of phosphate-buffered saline (PBS) containing 4 mM EDTA, 0.5% NP-40, and 1% 2-mercaptoethanol (final concentrations). Peptide N-glycosidase F (PNGase F; Cat# 11365177001, Roche Applied Science, Basel Switzerland) was added at a final concentration of 20 mU/μL, followed by incubation at 37°C for 16 h.
9
Mass Spectrometry Glycoproteomic Protocol
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Sodium borohydride, sodium chloride, Dowex cation-exchange resin (50W-X8), ammonium bicarbonate (ABC), TFA, Dulbecco’s PBS (DPBS), hydrochloric acid (HCl), and dl -DTT were purchased from Sigma–Aldrich. Ethanol (Reag. Ph. Eur) and bovine submaxillary mucin (BSM), type I-S, were purchased from Merck. Tandem mass tag (TMT)pro label reagents, 8 M guanidine hydrochloride, Dulbecco’s modified Eagle’s medium, 0.25% trypsin/EDTA, and fetal calf serum (FCS) were obtained from Thermo Fisher Scientific. Potassium hydroxide was obtained from Honeywell Fluka. Solid phase extraction bulk sorbent carbograph was obtained from Grace Discovery Sciences. HPLC SupraGradient acetonitrile (methyl cyanide [MeCN]) was obtained from Biosolve. Peptide N-glycosidase F (PNGase F) and complete EDTA-free protease inhibitor cocktail tablets were purchased from Roche Diagnostics. A 96-well PP filter plate was purchased from Orochem Technologies. MultiScreen HTS 96 multiwell plates (hydrophobic Immobilon-P polyvinylidene difluoride [PVDF] membrane) and 96-well PP microplate were obtained from Millipore.
10
Glycoprotein Characterization Protocol
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AA, AB, ABEE, AP, sodium cyanoborohydride, and pyridine-borane were purchased from Fujifilm Wako Pure Chemicals (Tokyo, Japan). AP was further purified by crystallization from hexane three times. A mixture of isomaltooligosaccharides (~20mers) was purchased from Seikagaku Kogyo (Tokyo, Japan). Bovine pancreas ribonuclease B and fetal calf serum fetuin were purchased from Sigma-Aldrich, Japan (Tokyo, Japan). Peptide N-glycosidase F (PNGase F) was purchased from F. Hoffmann-La Roche (Mannheim, Germany). Sephadex G15 fine and Sephadex LH-20 were obtained from GE Healthcare, Japan (Tokyo, Japan). All other solvents and reagents were of analytical grade.
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