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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)
- 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!)
- Mix, and rinse syringe once with mixture
- Place samples in ethanol/dry ice bath and allow to cool for 20 seconds
- Add 50mL TFMS/toluene mix to each sample by letting it run down side of vial over 15-20 seconds.
- Place vials at -20°C for 5 minutes
- After 5 minutes, shake briefly to aid melting of contents and solvation of sample
- Shake after another 5 minutes
- Leave vial in freezer for 4 hours
Neutralisation of Excess TFMS
Prepare pyridine solution: pyridine:methanol:water at ratio of 3:1:1
- Remove samples from freezer and remove caps and septa (neutralisation is highly exothermic so this allows gas to escape)
- Place vials in ethanol/dry ice bath and leave for 20 seconds
- 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
- Transfer vial to dry ice for 5 minutes
- Transfer to wet ice for 15 minutes
- Transfer sample to 2mL eppendorf tube
- 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
Use this procedure if precipitate formed during neutralisation.
- Cool sample to 4°C and incubate for 30 minutes
- Centrifuge at max speed for 15 mins at 4°C
- Keep supernatant in case not all has precipitated
- Resuspend pellet in cold buffer(?) and spin
- Repeat at least 3 more times.
Keep all supernatants, pool, evaporate and reconstitute with buffer. Isolate remaining peptide using dialysis or gel filtration
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Posted By: ARGERINE on 7/6/2011 10:28:50 AM