Enzymatic CarboRelease Kit

Kit Capacity
The quantity of enzymes recommended in the protocols is sufficient to deglycosylate approximately 100 μg of an average glycoprotein in the time given. PNGase F cleavage is generally the rate limiting reaction due to the slow removal of some sterically hindered N- linked residues, even when the glycoprotein is denatured. Since all of the enzymes retain activity under reaction conditions for several days, a much larger quantity of glycoprotein may be deglycosylated if incubation is extended. Conversely, there is no need to use the recommended amounts of enzymes if quantities much less than 100 μg of glycoprotein are being cleaved. The enzymes can be diluted into 1X Reaction Buffer 7. They will remain stable in diluted form at 4°C.

Bovine Fetuin Control Protein
Bovine Fetuin contains sialylated N- and O- linked oligosaccharides13.
NOTE: Commercial preparations of Fetuin contain proteases which will eventually degrade the protein. The Fetuin Control has been heat treated at 90°C for 10 minutes to inactivate the proteases. The Fetuin solution can be stored at 4°C.

Denaturing Protocol
1. Dissolve 100 μg or less of a glycoprotein in 30 μL deionized water in an Eppendorf tube.
2. Add 10 μL 5X Reaction Buffer 7 and 2.5 μL Denaturation Solution. Mix gently.
3. Heat at 100°C for 5 minutes.
NOTE: Some proteins may precipitate when heated with SDS. In this event, omit the heat treatment and increase the incubation time to 24 hours after adding enzymes.
4. Cool to room temperature. Add 2.5 μl Triton X-100 solution. Mix gently.
NOTE: Failure to add Triton X-100 may result in the reduction of activity of some enzymes.
5. Add 1 μL each of each enzyme.
6. Incubate for 3 hours at 37°C.
7. Analyze by method of choice.

Alternatively, the enzymes may be added individually or sequentially in order to determine what types of oligosaccharides are present on the glycoprotein.

Non-denaturing Protocol
1. Dissolve 100 μg or less of a glycoprotein in 35 μL deionized water in an Eppendorf tube.
2. Add 10 μL 5X Reaction Buffer 7.
3. Add 1 μL of each enzyme.
4. Incubate for 24 hours at 37°C.

An aliquot should be deglycosylated using the denaturing protocol to provide a gel standard for the fully deglycosylated protein. The position of the native protein can then be compared with this standard to judge the extent of deglycosylation.

References to Deglycosylate Glycoproteins

Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100:1- 14 (1979).

Kim MS, Leahy D. Enzymatic deglycosylation of glycoproteins. Methods Enzymol.533:259-63 (2013).

Magnelli PE, Bielik AM, Guthrie EP. Identification and characterization of protein glycosylation using specific endo- and exoglycosidases. J Vis Exp. Dec 26;(58):e3749 (2011).

Segu ZM, Hussein A, Novotny MV, Mechref Y. Assigning N-glycosylation sites of glycoproteins using LC/MSMS in conjunction with endo-M/exoglycosidase mixture. J Proteome Res. Jul 2;9(7):3598-607 (2010).

Sojar, H. T. and O.P. Bahl. A chemical method for the deglycosylation of proteins. Arch Biochem Biophys 259:52-57 (1987).

Tarentino A.L. and T.H. Plummer. Enzymatic deglycosylation of asparagine- linked glycans: purification, properties, and specificitly of oligosaccharide- cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymol 230: 44-57 (1994).

Uchida, Y., Y. Tsukada and T. Sugimori. Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J Biochem (Tokyo) 86:573-585 (1979).

Zhang W, Wang H, Zhang L, Yao J, Yang P. Large-scale assignment of N-glycosylation sites using complementary enzymatic deglycosylation. Talanta. Jul 15;85(1):499-505 (2011).