Deglycosylatio using TFMS

 Deglycosylation Using TFMS

Glycosylation is one of the most common posttranslational modifications of proteins. Glycans attach to the peptide backbone through either amide or glycosidic bonds (N-linked or O-linked) and may be removed either enzymatically or chemically. Enzymatic modes offer mild conditions, but are specific for either N- or O-linked glycans. Chemical methods though are non-specific, but capable of removing the entire glycan complement under appropriate reaction conditions. TFMS is a strong acid, achieves rapid, non-selective deglycosylation without compromise of the protein moiety, permitting subsequent analysis for proteomics and many other downstream analytical applications 

Deglycosylation Using TFMS

(Perform in a Fume Hood) 

1. Open TFMS and add toluene to final concentration of 10% (transfer 180mL TFMS to vial and add 20mL toluene) (aliquot remaining TFMS into glass vials – after finish adding mix to samples!)
2. Mix, and rinse syringe once with mixture
3. Place samples in ethanol/dry ice bath and allow to cool for 20 seconds
4. Add 50mL TFMS/toluene mix to each sample by letting it run down side of vial over 15-20 seconds.
5. Place vials at -20°C  for 5 minutes
6. After 5 minutes, shake briefly to aid melting of contents and solvation of sample
7. Shake after another 5 minutes
8. Leave vial in freezer for 4 hours

Neutralisation of Excess TFMS

Prepare pyridine solution: pyridine:methanol:water at ratio of 3:1:1

1. Remove samples from freezer and remove caps and septa (neutralisation is highly exothermic so this allows gas to escape)
2. Place vials in ethanol/dry ice bath and leave for 20 seconds
3. Using pipette, add 150mL of pyridine solution by letting it flow down side of vial over 15-20 seconds. Leave in bath for at least another 20 seconds
4. Transfer vial to dry ice for 5 minutes
5. Transfer to wet ice for 15 minutes
6. Transfer sample to 2mL eppendorf tube
7. Add 400mL of neutralisation solution (if solution left in glass vial, rinse and transfer to 2mL tube) and mix briefly (0.5% ammonium bicarbonate)

Here the polypeptide may form a precipitate

 Recovery of deglycosylated polypeptide

 Precipitation

Use this procedure if precipitate formed during neutralisation.

1. Cool sample to 4°C and incubate for 30 minutes
2. Centrifuge at max speed for 15 mins at 4°C
3. Keep supernatant in case not all has precipitated
4. Resuspend pellet in cold buffer(?) and spin
5. Repeat at least 3 more times.

Keep all supernatants, pool, evaporate and reconstitute with buffer. Isolate remaining peptide using dialysis or gel filtration