Aliquots (150 μg) of mouse monoclonal anti-MBP antibody (New England Biolabs), human or rabbit polyclonal IgG (Sigma-Aldrich) were incubated with Rapid PNGase F (New England Biolabs) in a total volume of 50 μL of Rapid PNGase F reaction buffer for 10 min at 50 °C. For use in lectin ELISA experiments, the deglycosylated antibodies were dialyzed against 20 mM sodium phosphate, pH 7.4, 150 mM NaCl using 10,000 Da MWCO dialysis membranes. For glycan labeling, the liberated N-glycans were cleaned and desalted using SPE Supelclean columns ENVI-18 100 mg and ENVI-CARB 100 mg (Supelco).
Rapid pngase f
Manufactured by New England Biolabs
Sourced in United States
Rapid PNGase F is an enzyme from New England Biolabs used for the deglycosylation of glycoproteins. It is a recombinant version of the Peptide-N-Glycosidase F enzyme, which cleaves the bond between the asparagine residue and the N-linked glycan.
Automatically generated - may contain errors
Lab products found in correlation
Rapid pngase f buffer, by New England Biolabs (3 mentions)
Ammonium formate, by Avantor (2 mentions)
Discovery glycan solid phase extraction tubes, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Xbridge protein beh c4 column, by Waters Corporation (1 mentions)
Acquity uplc h class, by Waters Corporation (1 mentions)
Unifi software version 1, by Waters Corporation (1 mentions)
Xevo g2 xs qtof, by Waters Corporation (1 mentions)
Trypsin, by New England Biolabs (1 mentions)
Trypsin, by Merck Group (1 mentions)
Iodoacetamide iaa, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Lct premier xe, by Waters Corporation (1 mentions)
Nanoacquity pump, by Waters Corporation (1 mentions)
Bovine fetuin, by Merck Group (1 mentions)
Sepharose cl 4b, by Merck Group (1 mentions)
Lc ms grade ammonium formate, by Merck Group (1 mentions)
Pa glucose oligomer, by Takara Bio (1 mentions)
Ribonuclease b rnase b, by Merck Group (1 mentions)
Blotglyco kit, by Sumitomo Bakelite (1 mentions)
16 protocols using rapid pngase f
1
Deglycosylation of Antibodies for Lectin ELISA
2
Deglycosylation of Worm Proteins
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Protein extraction from worms and immunoprecipitation were carried out as described previously (Hisamoto et al. 2019 (link)). For PNGase treatment, the bead pellets were further washed with wash buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA) for several times and then treated with Rapid PNGase F (New England Biolabs) according to the instruction manual. The treated or untreated samples were boiled with SDS sample buffer and subjected to immunoblotting as described previously (Li et al. 2012 (link); Hisamoto et al. 2018 (link)).
3
Panitumumab Tetrazine Conjugation Protocol
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Panitumumab (PAN, 20 mg/mL, PBS buffer pH 7.4) was buffer-exchanged to 5 mg/mL and reacted with 5 eq excess of the tetrazine NHS ester (CAS#1244060-64-9, 5 mg/mL in DMSO) at 4 °C overnight. The conjugate was purified with 0.5 M HEPES buffer (pH 7.2) using a PD-10 column. The purity of the conjugate (PAN-Tz) was confirmed by analytical size-exclusion HPLC. The protein concentration was determined by BCA assay. The tetrazine to antibody ratio was 4.5 ± 0.2, as determined by Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-ESI-MS) using an Acquity UPLC H-Class coupled to a Xevo G2-XS QTof (Waters Corporation, Milford, MA, USA). The sample was de-glycosylated using Rapid PNGase F (New England BioLabs, Ipswich, MA, USA) before analysis and desalted online using an XBridge Protein BEH C4 column (Waters Corporation) with a water/acetonitrile gradient containing 0.1% formic acid. Data processing was performed using UNIFI software version 1.9.4.053 (Waters Corporation) and peaks were assigned based on the unmodified antibody as a reference.
4
RapiFluor-MS Glycan Labeling Optimization
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Optimization experiments were performed with Herceptin to determine RapiFluor-MS labelling efficiency as glycoprotein mass was increased past the recommended 15 µg limit per reaction (Supplementary Fig. 8 ), as stipulated in the documentation included with Glycoworks RapiFluor-MS N-glycan basic kit (Waters). For each labelling reaction, a total of 150 µg of Endo-H pre-treated (glyco)peptides containing only complex N-glycans were reacted with Rapid PNGase-F (New England Biolabs) as described elsewhere36 (link). Liberated glycosylamines were reacted with RapiFluor-MS, then desalted on PD Minitrap G-10 columns. RapiFluor-MS-labelled glycans from 8 reactions were pooled and dried in a centrifugal evaporator for mass spectrometry analysis.
5
Membrane Protein Extraction and Glycan Release
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A sample of membrane protein extraction from cells (100–200 μg of protein) was added at 1/10 of the sample volume of 1 M ammonium bicarbonate (Sigma-Aldrich) and 5 μL of 0.2 M dithiothreitol (DTT, Sigma-Aldrich). The sample mixture was reduced by DTT at 60 °C for 30 min followed by alkylation with 5 μL of 0.5 M iodoacetamide (IAA, Sigma-Aldrich) by incubation in the dark at room temperature for 30 min. Then, the sample mixture was incubated at room temperature with 1.25 μL of 0.2 M DTT to remove excess IAA. The sample mixture was then treated with 5 μL of 200 ng/μL trypsin (Sigma-Aldrich) at 37 °C for at least 5 h with shaking (1000 rpm), followed by heat inactivation of the enzyme at 100 °C for 5 min. N-Glycans were enzymatically released from trypsin-digested glycopeptides by incubation with 1 μL Rapid PNGase F (New England Biolabs, Ipswich, MA, USA) at 37 °C for at least 16 h with shaking (1000 rpm).
6
Mass Spectrometry Analysis of Deglycosylated Proteins
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For mass spectrometry analysis, a Waters (Baden, Switzerland) nanoAcquity pump equipped with Waters LCT Premier XE and a Acquity UPLC Protein C4 column, 300 Å, 1.7 μm, 1 x 50 mm was used in ESI positive mode at a flow rate of 70 μL/min. Mobile phases used were (A) water/acetonitrile 9:1 with 0.1% trifluoroacetic acid and (B) acetonitrile/water 9:1 with 0.1% trifluoroacetic acid, from 10% (B) to 65% (B) within 10 minutes. Proteins have been analysed (if not otherwise mentioned) reduced and deglycosylated by the use of Rapid PNGase F (New England Biolabs, Ipswich, MA, USA) according to the following representative procedure: 5.7 μL miliQ water was added to 10.3 μL (12 μg) protein solution. 4 μL Rapid PNGase F buffer, containing dithiothreitol (DTT) for mild reduction of disulfide bonds (5x) and 1 μL Rapid PNGase F [18 (link)] for deglycosylation was added. The mixture was incubated at 50°C for 15 minutes.
7
Comprehensive Glycoprotein Analysis Reagents
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Bevacizumab (Avastin) and
trastuzumab (Herceptin)
were purchased from Chugai Pharmaceutical (Tokyo, Japan). Nivolumab
(Opdivo) was purchased from Ono Pharmaceutical (Osaka, Japan), and
ramucirumab (Cyramza) was purchased from Eli Lilly Japan (Hyogo, Japan).
Rituximab (Rituxan) was obtained from Zenyaku Kogyo (Tokyo, Japan).
Rapid PNGase F was purchased from New England Biolabs (Tokyo, Japan).
The BlotGlyco kit was from Sumitomo Bakelite Co. Ltd. (Tokyo, Japan),
and PA-glucose oligomer was obtained from TaKaRa (Kyoto, Japan). Sepharose
CL-4B, ribonuclease B (RNase B), bovine fetuin, and LC/MS-grade ammonium
formate were obtained from Sigma-Aldrich (St. Louis, MO). All other
unspecified reagents were products of FUJIFILM Wako Pure Chemical
Corporation (Osaka, Japan).
trastuzumab (Herceptin)
were purchased from Chugai Pharmaceutical (Tokyo, Japan). Nivolumab
(Opdivo) was purchased from Ono Pharmaceutical (Osaka, Japan), and
ramucirumab (Cyramza) was purchased from Eli Lilly Japan (Hyogo, Japan).
Rituximab (Rituxan) was obtained from Zenyaku Kogyo (Tokyo, Japan).
Rapid PNGase F was purchased from New England Biolabs (Tokyo, Japan).
The BlotGlyco kit was from Sumitomo Bakelite Co. Ltd. (Tokyo, Japan),
and PA-glucose oligomer was obtained from TaKaRa (Kyoto, Japan). Sepharose
CL-4B, ribonuclease B (RNase B), bovine fetuin, and LC/MS-grade ammonium
formate were obtained from Sigma-Aldrich (St. Louis, MO). All other
unspecified reagents were products of FUJIFILM Wako Pure Chemical
Corporation (Osaka, Japan).
8
Glycan Analysis by Mass Spectrometry
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A series of Remab6 (~ 25 μg) was treated with Rapid™ PNGase F (Cat#P0710S, New England Biolabs, Inc.) for 45 min at 50 °C, and released N-glycans were separated from peptides via C18 Sep-Pak pre-packed columns (Cat#WAT054945, Waters Corp.), followed by lyophilization. All glycans were permethylated, extracted with chloroform and purified by C18 Sep-Pak pre-packed columns prior to mass spectrometric analysis with an ultraXtreme mass spectrometer (Bruker Corp.). Data was acquired under positive mode via flexControl (Bruker Daltonics, version 3.4, build 135) and further processed with flexAnalysis (Bruker Daltonics, version 3.4, build 76). Each peak was analyzed and annotated manually with the aid of GlycoWorkBench.
9
Fluorescent Labeling and Separation of HIV gp145 N-Glycans
10
Antibody Deglycosylation and Stability
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Deglycosylated antibody was prepared by incubating 10 μg of a commercially available anti-hAFP antibody (#MIA1305, Thermo Fisher Scientific) with 100 U of PNGase-F (NEB) in Rapid PNGase-F (non-reducing format) buffer for 15 min at 50 °C. Time- and temperature-dependent protein stability was measured by incubating glycosylated, deglycosylated, or aglycosylated antibodies in PBS buffer at either 4 or 37 °C for up to 14 days. The incubated antibodies were run on 4–20% Mini-Protein TGXTM gels (Bio-Rad) and visualized by Coomassie blue staining. The protein stability was also monitored by incubating antibodies in 0.1 M phosphate buffer adjusted to pH 3.0, 7.0, or 10.0 for 0–14 days. Alternatively, each antibody was incubated in PBS buffer containing 0–3% H2O2 at 37 °C for 5 h. The integrity of the antibodies was measured by the ELISA described above.
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