Goal:
To prepare serum for N-glycan isolation by Rapid PNGaseF and analysis by mass spectrometry.
Notes:
Solvents are HPLC grade or higher.
Protocol:
- Lyophilize 5 to 10 µl of serum (achieve in less than an hour) in a 500 µl tube or in a 200 µl PCR tube if using a PCR thermocycler for buffer and enzyme incubations.
- Resuspend the dried material thoroughly in 16 µl of MilliQ Water, add 4 µl of Rapid PNGaseF buffer (New England Biolabs, #P0710S), and incubate at 70°C for 15 min.
- Cool down the sample for a few minutes and add 1 µl of Rapid PNGaseF, incubate at 50°C for 30 to 60 min.
- Condition a C18 Sep-Pak (50 mg) column (Waters, #WAT054955) with methanol, 5% acetic acid (Fisher, #A38-212), 1-propanol and 5% acetic acid in succession. Add 100 µl 5% acetic acid to the Rapid PNGaseF-treated serum sample, pipet to resuspend any precipitate and load the mixture onto the C18 column. Collect the flow through into a 1.5 ml tube.
- Add another 100 µl of 5% acetic acid to the tube used to incubate the serum sample and Rapid PNGaseF, rinse the walls of tube and load onto the C18 column. Wash the column with 1 ml of 5% acetic acid. Collect these fractions in the same 1.5 ml tube.
- Lyophilize the sample and proceed to permethylation (Small Scale permethylation protocol).