Each of 4 peptides-fractions (for human milk glycolproteomics) was dissolved in 40 μL enrichment buffer solution (80% acetonitrile/1% trifluoroacetic acid), and transferred to a hydrophilic microcolumn (HILIC column, Dalian Institute of Chemistry Physics, Chinese Academy of Sciences, China) (13 (link)), and the enrichment was completed by centrifugation at 4000 g for 15 min. The microcolumns were then washed 3 times with enrichment buffer. The glycopeptides were then eluted with 10% acetonitrile and lyophilized. Then, the lyophilized glycopeptides were reconstituted in 50 μL of 50 mM ammonium bicarbonate buffer dissolved in heavy oxygen water (H218O), 2 μL PNGase F glycosidase (New England Biolabs, United Kingdom) was added, and the peptides were digested overnight at 37°C. Finally, the salt was removed according to the instructions of C18 ZipTips (Sigma, United States), and the samples were freeze-dried for LC/MS analysis.
Pngase f glycosidase
Manufactured by New England Biolabs
Sourced in United Kingdom
PNGase F glycosidase is an enzyme that cleaves the linkage between the asparagine residue and the carbohydrate in N-linked glycoproteins. It is commonly used in the analysis of glycoproteins.
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5 protocols using pngase f glycosidase
1
Glycopeptide Enrichment and Analysis
2
N-Glycan Release and Purification
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The N-glycans released by PNGase F glycosidase (New England Biolabs, Beverly, MA) according to previous protocols.26 (link) Briefly, 200 μg glycoproteins were concentrated and desalted by adding to a 3K centrifugal ultrafiltration. Then, the obtained glycoproteins were denatured with 8 M urea, 10 mM DTT and 10 mM IAM, and exchanged buffer into 40 mM NH4HCO3 by using 3K centrifugal ultrafiltration. After that, 2 μg trypsin was added to digest the glycoproteins overnight at 37 °C. The mixture was heated at 80 °C for 5 min to deactivate the activity of trypsin. After the temperature of mixture restored to room temperature, 2 μL PNGase F was added and incubated at 37 °C overnight to release the N-linked glycans from the glycopeptides. Finally, the filtrates that contained peptides and N-linked glycans were collected by centrifugation, and the released glycans were purified with HyperSep Hypercarb SPE cartridges (25 mg, 1 mL; Thermo Scientific) according to the manufacturer's recommendation. The glycans were eluted by 0.5 mL of elution solution (50% (v/v) acetonitrile with 0.1% (v/v) TFA). The purified glycans were collected and lyophilized.
3
Efficient N-Glycan Release and Purification
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The N-glycans were released by PNGase F glycosidase (New England Biolabs, Beverly, MA) according to previous protocols (25 (link), 26 (link)). Briefly, 200 μg of isolated glycoproteins were concentrated and desalted by adding to a size-exclusion spin ultrafiltration (Amicon Ultra-0.5 mL 10,000 MW cut off, Millipore). Then, the obtained glycoproteins were denatured with 8M urea (Sigma-aldrich), 10 mM DTT, and 10 mM IAM (Sigma-Aldrich). Followed by the exchange of buffer into 40 mM NH4HCO3 via using 10 K centrifugal ultrafiltration. After that, 5 μL of PNGase F (NEB) were added into ultrafiltration and incubated over night with shaking at 37°C. The reaction was stopped by incubating the mixture at 80°C for 5 min. After centrifuge, the released N-glycans were collected and purified with HyperSep Hypercarb SPE cartridges (25 mg, 1 mL; Thermo Scientific) according to the manufacturer recommendation. The N-glycans were eluted by 0.5 mL of elution solution (50% (v/v) acetonitrile with 0.1% (v/v) TFA). The purified N-glycans were collected and lyophilized.
4
Glycoprotein Deglycosylation with PNGase F
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Following the manufacturer’s instruction of glycoprotein treatment with PNGase F glycosidase (NEB Inc., #P0704), 3 μg of recombinant hRNase 1 protein (Sino Biological Inc., #13468-H08H) was combined with 1 μl of 10× Glycoprotein Denaturing Buffer and water to make up a 10 μl total reaction volume. The mixture was denatured by heating at 100 °C for 10 min and chilled on ice for 2 min, and 2 μl of 10× GlycoBuffer 2, 2 μl of 10% Nonidet P-40, and 6 μl of water were then added to make up a 20 μl total reaction volume. The mixture was then incubated at 37 °C overnight with or without 1 μl of PNGase F to keep the final glycerol concentration equal to 5% and subjected to in vitro binding assay.
5
N-Glycan Release and Purification
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N-Glycans were released by PNGase F glycosidase (New England Biolabs, Beverly, MA) according to previous protocols.32,34 (link) Briefly, the derivatized glycoproteins were denatured with 8 M urea (Sigma-Aldrich), 10 mM DTT (dithiothreitol, Sigma-Aldrich) and 10 mM IAM (iodoacetamide, Sigma-Aldrich), and exchanged buffer into 40 mM NH4HCO3 by 10 kDa centrifugal ultrafiltration. After that, 2 μL of PNGase F (NEB) was added and incubated with shaking at 37 °C overnight to release the N-linked glycans. The reaction was terminated by incubating the mixture at 80 °C for 5 min. By centrifuging at 12 000 × g for 10 min, the mixture of N-linked glycans was collected. This eluting step was replicated twice with 40 mM NH4HCO3, and N-glycans were then evaporated to dryness using a vacuum centrifuge.
+ Expand
N-Glycans were released by PNGase F glycosidase (New England Biolabs, Beverly, MA) according to previous protocols.32,34 (link) Briefly, the derivatized glycoproteins were denatured with 8 M urea (Sigma-Aldrich), 10 mM DTT (dithiothreitol, Sigma-Aldrich) and 10 mM IAM (iodoacetamide, Sigma-Aldrich), and exchanged buffer into 40 mM NH4HCO3 by 10 kDa centrifugal ultrafiltration. After that, 2 μL of PNGase F (NEB) was added and incubated with shaking at 37 °C overnight to release the N-linked glycans. The reaction was terminated by incubating the mixture at 80 °C for 5 min. By centrifuging at 12 000 × g for 10 min, the mixture of N-linked glycans was collected. This eluting step was replicated twice with 40 mM NH4HCO3, and N-glycans were then evaporated to dryness using a vacuum centrifuge.
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