New England Biolabs, P6044S, Protein Deglycosylation Mix II

The Protein Deglycosylation Mix II contains all of the enzymes, reagents, and controls, to remove all N-linked, as well as many common O-linked glycans, from glycoproteins. Following the deglycosylation reaction, samples are ready to be prepared for mass spectrometry analysis Related Categories Exoglycosidases,, Endoglycosidases,, Proteome Analysis Applications Expression Systems,, Glycan Sequencing,, Protein Digestion, Specification Storage Buffer 50 mM NaCl 20 mM Tris-HCl 2.6 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: What is the difference between the discontinued Protein Deglycosylation Mix (NEB# P6039) and the new Protein Deglycosylation Mix II (NEB# P6044)? A: The Protein Deglycosylation Mix II (NEB# P6044) is a new and improved reformulation of the recently discontinued Protein Deglycosylation Mix (P6039). The Protein Deglycosylation Mix II is now made up of five recombinant, and highly pure enzymes that have been optimized for simultaneous deglycosylation of N- and O-linked glycans. The exact formulation change between the two products is shown in the table below: Protein Deglycosylation Mix (P6039) discontinued Protein Deglycosylation Mix II (P6044) PNGase F (Glycerol free) (P0705) PNGase F (Glycerol free), Recombinant (P0709) O-Glycosidase (P0733) O-Glycosidase (P0733) α2-3,6,8 Neuraminidase (P0720) α2-3,6,8,9 Neuraminidase A (P0722) β1-4 Galactosidase (P0730) β1-4 Galactosidase S (P0745) β-N-Acetylglucosaminidase (P0732) β-N-Acetylhexosaminidasef (P0721) Q: Two different reaction buffers (10X Deglycosylation Mix Buffer 1 and 2) are supplied with the Protein Deglycosylation Mix II. How do I know which buffer to use? A: The 10X Deglycosylation Mix Buffer 1 (B6044S) should be used when native (non-denaturing) conditions are necessary. 10X Deglycosylation Mix Buffer 1 is 500mM sodium phosphate, pH 7.5. This buffer is mild and contains no denaturing or reducing agents. In this buffer system, the glycoprotein will remain 100% intact. However, deglycosylation under native conditions typically requires excess enzyme and longer incubation times, and in some cases glycan removal will not be complete. Therefore, when denaturation is acceptable, we recommend using the 10X Deglycosylation Mix Buffer 2 (B6045S). This buffer will reduce the glycoprotein, but will also provide the most efficient and complete level of deglycosylation. 10X Deglycosylation Mix Buffer 2 is mass spectrometry compatible and does not contain SDS. Reference the “Protocols and Manuals” tab for the specific protocol associated with each buffer system. Q: Are downstream analysis such as HPLC and Mass Spectrometry compatible with the Protein Deglycosylation Mix II? A: Yes, all of the reagents included in the Protein Deglycosylation Mix II are compatible with both HPLC and Mass Spectrometry applications. To prepare samples for liquid chromatography and/or mass spectrometry, the target protein can be prepared using micro-dialysis or micro-filtration (refer to the “Glycoproteomics: Buffer Exchange Protocols” ). Q: I tried the Protein Deglycosylation Mix II on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem? A: The Protein Deglycosylation Mix II includes the enzymes necessary to remove carbohydrates attached to Asparagine residues, simple core 1 and core 3 O-linked carbohydrates attached to Ser/Thr, and decorated core 1 and 3 O-glycans (with lactosamine extensions). There is the possibility that the carbohydrate may be resistant to PNGase F (a rare occurrence). This happens when the core N-Acetylglucosamine is modified by an α1-3Fucose (often found in plant or insect proteins). Also, O-glycans extended with fucose, alpha-galactose, and other residues will be resistant to this cocktail, unless additional exoglycosidase enzymes are added. If possible, use 10X Deglycosylation Mix Buffer 2 to reduce the protein prior to deglycosylation (this buffer is compatible with sample preparation for mass spectrometry). The secondary and tertiary structure of proteins can prevent endoglycosidases from reaching their substrate, thus making reduction a crucial step in efficient cleavage. If you do not want to reduce then consider adding more enzyme and using longer incubation times. The sample itself could cause enzyme inactivation. Avoid sample buffers containing SDS as this detergent inhibits PNGase F, O-glycosidase, and β1-4 Galactosidase. The sample could also cause a drop (or rise) in pH, particularly if large volumes are used (in those cases, it would be ideal to exchange the sample in a low molarity, neutral buffer). Confirm that your target protein should be glycosylated. Some glycosylation predictors might annotate sites that are not occupied in nature. Also, glycosylation patterns change among different tissues, organisms, and/or growth stages. Likewise, different expression systems might result in proteins with unexpected changes in glycosylation. Finally, the shift in mobility of your protein (SDS-PAGE, Western blot) may be difficult to visualize (particularly when a small carbohydrate has been removed from a large protein). If possible run side by side negative controls. Optimize loading and running conditions to facilitate the detection of subtle mass changes. Q: How much Protein Deglycosylation Mix II should I use to remove the carbohydrates under native (non-denaturing) conditions? A: When the protein is not reduced or denatured, the Protein Deglycosylation Mix II may have a more difficult time reaching the cleavage site of the carbohydrate (because of the secondary and tertiary protein structure). Under reducing conditions, we recommended using 5 μL of Deglycosylation Mix per 100 μg of protein. Under native conditions it is recommended to use extra Deglycosylation Mix and extended incubation times. Some complex proteins may only result in partial deglycosylated under native conditions, even after long incubations. The exact amount of Deglycosylation Mix required may be specific to each protein’s level of complexity and must be determined empirically. Q: What is a good control substrate for the Protein Deglycosylation Mix II? A: We suggest Fetuin (NEB# P6042), because it is a glycoprotein containing sialylated N-linked and O-linked glycans. A 500μg vial of Fetuin is supplied with the Protein Deglycosylation Mix II. Q: Are Protease Inhibitors acceptable for use with the Protein Deglycosylation Mix II reaction? A: When a protein is reduced or denatured it is more susceptible to cleavage by proteases. For this reason a protease cocktail containing the following can be used with the Protein Deglycosylation Mix II protocol: Aprotinin (10 μg/ml final concentration) Benzamidine (1mM final concentration) Pepstatin (10 μg/ml final concentration) Leupeptin (1μM final concentration) EGTA (1 mM final concentration) EDTA (1 mM final concentration) PMSF (1 mM final concentration) Make a 1000X concentrated stock of each inhibitor in water; except Pepstatin which should be dissolved in methanol. Note: PMSF has the ability to modify basic residues on glycoprotein substrates.