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Cell N-glycan preparation for MS analysis

Goal:

To prepare cells for N-glycan isolation and analysis by mass spectrometry.

Notes:

Glassware and glass tubes are cleaned with MilliQ water and dried beforehand.
Reagents are weighted on aluminum foil with utensils cleaned with MilliQ water beforehand. Whenever possible, liquid reagents are handled with disposable glass pipettes.
Solvents are HPLC grade or higher.

Protocol:

  1. Thaw 5x106 cells (2 to 10x106 cells) on ice for 30 min or more. Add 1 to 2 ml of ice-cold lysis buffer: 25 mM TRIS, 150 mM NaCl, 5 mM EDTA and 0.5% (w/v) CHAPS, pH 7.4. Adjust the pH before adding the CHAPS. Resuspend the cells thoroughly.
  2. In lysis buffer, sonicate the sample at low intensity with 4 to 5 pulses of 10 sec each separated by 30-60 sec of pause intervals.
  3. Dialyze the sample against 50 mM ammonium bicarbonate at 4°C for 16h-24h, changing the buffer 3 times. The molecular cut-off should be between 1 and 5 kDa. After dialysis, transfer the sample into a 15 ml tube and lyophilize the sample.
  4. Prepare a fresh 2 mg/ml solution of 1,4-dithiothreitol (DTT, Sigma, #10197777001) in 0.6 M TRIS buffer pH 8.5. Resuspend the dry sample thoroughly in 1 ml of the DTT solution and incubate at 50°C for 1.5 hours.
  5. Prepare a fresh 12 mg/ml solution of iodoacetamide (IAA, Sigma, #I6125) in 0.6 M TRIS buffer pH 8.5. Add 1 ml of IAA solution to the DTT-treated sample and incubate at RT in the dark for 1.5 hours.
  6. Dialyze the sample against 50 mM ammonium bicarbonate at 4°C for 16h-24h, changing the buffer 3 times. The molecular cut-off should be between 1 and 5 kDa. After dialysis, transfer the sample into a 15 ml tube and lyophilize the sample.
  7. Resuspend the dry sample in 1 ml of a 500 µg/ml solution of TPCK-treated trypsin (Sigma, #4352157) in 50 mM ammonium bicarbonate and incubate overnight (12-16h) at 37°C. Stop the reaction by adding 2 drops of 5% acetic acid (Fisher, #A38-212).
  8. Condition a C18 Sep-Pak (200 mg) column (Waters, #WAT054945) with methanol (Fisher, #A452-4), 5% acetic acid, 1-propanol (Fisher, #A414-1) and 5% acetic acid. Load the trypsin-digested samples onto the C18 column.
  9. Wash the column with 6 ml of 5% acetic acid and elute the peptides from the C18 column with 2 ml of 20% 1-propanol, then 2 ml of 40% 1-propanol and finally 2 ml of 100% 1-propanol. Pool all of the eluted fractions and lyophilize the sample.
  10. Resuspend the dried material thoroughly in 200 µl of 50 mM ammonium bicarbonate and add 3 µl of PNGaseF (New England Biolabs, #P0704). Incubate at 37°C for 4h. Then add another 5 µl of PNGaseF for overnight (12-16h) incubation at 37°C. Stop the reaction by adding 2 drops of 5% acetic acid.
  11. Condition a C18 Sep-Pak (200 mg) column with methanol, 5% acetic acid, 1-propanol and 5% acetic acid in succession. Load the PNGaseF-digested sample onto the C18 column. Collect the flow through.
  12. Wash the column with 6 ml of 5% acetic acid and collect the wash fractions. Pool the flow through and wash fractions, lyophilize the sample and proceed to permethylation.