Purified mAb CO17 and mAbP COK were treated with pepsin to digest the protein into glycopeptides, and digested peptides were passed through a C18 sep-pak cartridge (Waters, Lexington, MA). The samples were washed with 5% acetic acid to remove contaminants such as salts and free glycans. The fraction was eluted in series solutions with 20, 40, and 100% iso-propanol, and the eluted fractions were dried using a speed vaccum dryer. To release N-glycans, PNGase A (Roche, Basel, Switzerland) was added to each sample and incubated overnight at 37°C. The released N-glycans were purified using a graphitized carbon resin (Alltech, Lexington, MA). Purified glycans were resolubilized in HPLC water, spotted onto a stainless steel test plate, and treated by 0.8 μL of 50 mg·mL-1 2,5-dihydroxybenzoic acid in 50% acetonitrile. The spot was rapidly dried under vacuum for more homogenous crystallization. Analysis was carried out by MALDI-TOF MS using a Bruker ultrafleXtreme™ system.
Pngase a
Manufactured by Roche
Sourced in Switzerland
PNGase A is a type of enzyme that is used in laboratory settings for the cleavage of N-glycosidic linkages between asparagine residues and carbohydrates in glycoproteins. It functions to remove the carbohydrate moieties from glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Anthranilic acid, by Merck Group (4 mentions)
Biogel p10, by Bio-Rad (4 mentions)
Trypsin, by Merck Group (4 mentions)
C18 bakerbond spe cartridges, by Avantor (4 mentions)
Ultraflex 2 mass spectrometer, by Bruker (4 mentions)
N hydroxysuccinimide activated sepharose, by GE Healthcare (3 mentions)
Extract clean carbo spe columns, by Avantor (3 mentions)
Sep pak c18 cartridge, by Waters Corporation (2 mentions)
Pngase f, by Roche (2 mentions)
Ultraflextreme system, by Bruker (1 mentions)
Peptide n glycosidase f, by Roche (1 mentions)
Supelclean envi carb, by Merck Group (1 mentions)
Peptide n glycosidase a, by Roche (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
10 protocols using pngase a
1
Glycopeptide Analysis by MALDI-TOF MS
2
N-Glycan Analysis of Purified BmVAL-1
3
Glycan Analysis by MALDI-TOF MS
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Purified recombinant protein samples were first digested into glycopeptides using pepsin, as previously described [26 (link),48 (link)]. From the glycopeptides, N-glycan was released using PNGase A (Roche, Basel, Switzerland). The released N-glycans were purified using graphitized carbon resin from Carbograph (Alltech). The purified glycans were re-dissolved in a mixture of 90 μL dimethyl sulfoxide (DMSO), 2.7 μL water, and 35 μL iodomethane for solid phase permethylation while using a spin column method [49 (link)]. The resulting permethylated glycans were mixed in equal volumes with 10 mg/mL 2,5-dihydroxybenzoic acid that was prepared in 1 mM of a sodium acetate solution. The mixture was applied onto a matrix-assisted laser-desorption-ionization (MALDI) MSP96 ground steel target plate and dried for MALDI-TOF mass spectrometry. All of the mass spectra were acquired at an acceleration voltage of 20 kV.
4
Glycomic Analysis of Insect Cell Lines
5
N-glycan Enrichment and Analysis
6
BmVAL-1 N-Glycan Analysis Protocol
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For N-glycan analysis, 1–2 μg of purified BmVAL-1 was reduced and denatured for 10 min at 95 °C in PBS containing 1.3% (w/v) SDS and 0.1% (v/v) β-mercaptoethanol. SDS was neutralized by adding 2% (v/v) NP-40 prior to overnight digestion at 37 °C with trypsin (Sigma-Aldrich, USA), immobilized to N-hydroxysuccinimide-activated sepharose (GE Healthcare). trypsin beads were removed from the digestion mix by centrifugation and the pH of the mix was adjusted to 5 using 1 M sodium acetate. PNGase A (0.5 mU; Roche, Switzerland) was used to release N-glycans from BmVAL-1 while incubating overnight at 37 °C. The incubation mixture was applied to C18 Bakerbond™ SPE cartridges (JT Baker, USA) and the N-glycans were extracted from the flow-through on Extract Clean™ Carbo SPE columns. Eluted N-glycans were labeled with anthranilic acid (Sigma-Aldrich) and desalted by hydrophilic interaction chromatography on Biogel P10 (BioRad, USA). Samples in 75% acetonitrile were mixed with 1 μl of matrix solution (20 mg/ml 2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid) and were dried under a stream of warm air. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra (MS) were obtained using an Ultraflex II mass spectrometer (Bruker Daltonics, USA).
7
N-Glycan Release and Purification
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The purified samples were incubated twice with 2 μL of pepsin in an incubator at 37°C for 12 h to digest the protein into glycopeptides. The digested glycopeptides were collected using a C18 sep-pak cartridge (Waters, Lexington, MA, USA). Briefly, samples were passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants such as salts and free sugar. The fraction containing peptides and glycopeptides was eluted in a series of solutions with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid, and 100% iso-propanol, and the eluted fractions were dried in a speed vacuum system. PNGase A (Roche, Basel, Switzerland) was added to the samples to release N-glycans, and the samples were incubated overnight at 37°C. The released N-glycans were purified from the samples by using a graphitized carbon resin from Carbograph (Alltech, Lexington, MA, USA).
8
N-Glycan Analysis of HpVAL-4 Protein
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For N-glycan analysis, 1–2 μg of purified HpVAL-4 was reduced and denatured for 10 min at 95 °C in PBS containing 1.3% (w/v) SDS and 0.1% (v/v) β-mercaptoethanol. SDS was neutralised by adding 2% (v/v) NP-40 prior to overnight digestion at 37 °C with trypsin (Sigma–Aldrich, USA) immobilised to N-hydroxysuccinimide-activated Sepharose (GE Healthcare). trypsin beads were removed from the digestion mix by centrifugation and the pH of the mix was adjusted to 5 using 1 M sodium acetate. PNGase A (0.5 mU; Roche, Switzerland) was used to release N-glycans from HpVAL-4 while incubating overnight at 37 °C. The incubation mixture was applied to C18 Bakerbond™ SPE cartridges (JT Baker, USA) and the N-glycans were extracted from the flow-through on Extract Clean™ Carbo SPE columns. Eluted N-glycans were labelled with anthranilic acid (Sigma–Aldrich) and desalted by hydrophilic interaction chromatography on Biogel P10 (BioRad). Samples in 75% acetonitrile were mixed with 1 μl of matrix solution (20 mg/ml of 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% (v/v) trifluoroacetic) and were dried under a stream of warm air. Matrix-assisted laser desorption/ionisation (MALDI) time-of-flight mass spectra (MS) were obtained using an Ultraflex II mass spectrometer (Bruker Daltonics, USA).
9
Enrichment and Deglycosylation of N-Glycopeptides
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The HILIC enrichment of N-glycopeptides was processed as described with minor modifications [37 (link),38 (link)]. Briefly, the peptide fractions were equilibrated in 40 μL loading buffer (80% acetonitrile/1% trifluoroacetic acid), then pipetted into a tip containing HILIC beads (4 μm, 100 Å) (Dalian Institute of Chemical Physics, Dalian, China). After centrifugation at 4,000 g for 15 minutes, the glycopeptides were retained in the tip. Then, the HILIC tip was washed with 40 μL of loading buffer three times to remove the residual non-glycopeptides by centrifugation. The enriched glycopeptides were then eluted with 80 μL H2O and lyophilized by vacuum centrifugation. Finally, two hundred units of PNGase F (New England Biolabs, Ipswich, England) and PNGase A (Roche, Basel, Switzerland) in 50 μL 40 mM NH4HCO3 dissolved in H218O was applied to the samples and incubated overnight at 37°C for deglycosylation.
10
N-Glycan Analysis of Purified HpVAL-4
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