Chemical deglycosylation
Simplified sample recovery
Chemical deglycosylation of glycoproteins requires hydrolyzing glycosidic bonds without hydrolyzing peptide bonds. Such hydrolysis requires superacids (i.e. acids stronger than anhydrous sulfuric). The two most common are hydrofluoric, which is volatile and etches glass and trifluoromethanesulfonic, which is less volatile, but viscous. Both acids readily take up water from moist air and must be kept as dry as possible. Superacids are generally mixed with ethers, such as anisole, or thioethers, such as dimethyl sulfide, which lower acidity and scavenge reactive electrophiles. Details of the "protection" aren't fully understood..
Method
1- Prepare a 5 mg/ml solution of glycoprotein in distilled water.
2- Place 100 ug sample in a 3 ml Pyrex tube and vacuum dry for 15-30 min. Sample must be completely dry. Note: Reaction must be performed in a fume hood. Wear protective goggles, lab coat and gloves. Use extreme care as trifluoromethanesulfonic acid (TFMSA) is 30 times stronger than concentrated sulfuric acid!
3- Place samples on ice and carefully add 50 ul of TFMSA and 1/10th to 1/2 volume of anisole.
4- Incubate 3 hr on ice.
5- Carefully neutralize TFMSA with 125 ul of N-ethylmorpholine (NEM). We usually add 50 ml, shake, cool on ice and then add the remainder.
6- Add 5-10 volumes of AR acetone. Cover with Parafilm and mix well. Note that the TFMSA-protein-NEM mixture is highly viscous and requires considerable shaking to dissolve in acetone.
7- Incubate suspension overnight at -20oC or 1 hr at -90oF.
8- Centrifuge sample 10 min at 7K to pellet protein. Discard the supernatant.
9- Dry pellets under vacuum for about 30 min.
10- Resuspend pellets in 100 ul of Laemmli cracking buffer containing 10mM dithiothreitol. Heat 2 min at 100oC.
11- Alkylate proteins with 25mM iodoacetamide for 10 min at 50oC.
12- Electrophorese 3-10 ul aliquots on SDS polyacrylamide gels.
Notes: For discussion of superacids see p280 of Mechanism and Theory in Organic Chemistry (3rd ed), Lowry and Richardson (1987) as well as the table on p297.
The review by Edge (Biochem J 376, 339-350 (2003)) shows that most common protein modifications resist superacid hydrolysis.
Solvent precipitation of protein should work in step 1.
I recommend doing a time series (step 4). Shorter incubations may suffice.
Adding a carrier (such as Ficoll) and briefer cooling should work at step 6.