To isolate O-glycan for mass spectrometry analysis.
Glassware and glass tubes are cleaned with Milli Q water and dried beforehand.
Reagents are weighted on aluminum foils with utensils clean with Milli Q water beforehand. Whenever possible, liquid reagents are handled with disposable glass pipettes.
Solvents are HPLC grade or higher.
The starting material in most cases is N-glycan-free (with PNGaseF or other methods) glycopeptides.
- Prepare a 0.1 M NaOH solution. This can be quickly made in a 50ml falcon tube by mixing 20 ml of MilliQ with one pellet of NaOH (Sigma-Aldrich, #S8045).
- Add 1 ml of 0.1 M NaOH to 55 mg of NaBH4 (sodium borohydride, Sigma-Aldrich, #213462) in a clean glass tube, mix well.
- Add 400 µl of the borohydride solution to a lyophilized sample in a screw-capped glass tube and close the tube.
- Incubate at 45°C overnight (14-16 hours).
- Terminate the reaction by adding 4-6 drops of pure (100%) acetic acid (Fisher, #A414-1) or until fizzing stops.
- Prepare the Dowex ion exchange resin. A stock solution of Dowex 50W X8 (mesh size 200-400) (Sigma-Aldrich, #44519) is made by washing three times 100 g of resin with 100 ml of 4 M HCl (hydrochloric acid, Fisher A144SI-212). Wash the resin with 300 ml of MilliQ water and repeat this wash step ~15 times until the pH remains stable. Then wash the resin with 200 ml of 5% acetic acid three times. This stock solution is table for up to one year stored at 4°C.
- Assemble a desalting column with 2-3 ml of the Dowex resin prepared above in a small glass column (such as a glass Pasteur pipette). Wash the column with 10 ml of 5% acetic acid.
- Load the acetic acid-neutralized sample onto the column and wash with 3 ml of 5% acetic acid. Collect and pool flow through and wash.
- Lyophilize the collected material.
- Add 1 ml of acetic acid: methanol (Fisher, #A452-4) solution (1:9; v/v=10%) to the lyophilized sample. Vortex thoroughly and dry under a stream of nitrogen. Repeat this co-evaporation step three more times.
- Condition a C18 Spe-Pak column (column size depends on the sample) with methanol, 5% acetic acid, 1-propanol (Fisher, #A414-1) and 5% acetic acid.
- Resuspend the dried sample in 200 µl of 50% methanol and load onto the conditioned C18 column. Wash the column with 4 ml of 5% acetic acid. Collect and pool flow through and wash.
- Lyophilize the sample and proceed to permethylation.