To prepare in-gel glycoproteins for N-glycan isolation and analysis by mass spectrometry.
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.
- Samples are usually run on a standard SDS-page gel and bands visualized by Coomassie Blue staining.
- Excise and dice the gel band in small pieces. Transfer the gel pieces in a clean, low-binding 1.5ml plastic tube.
- Add 200 µl of a freshly prepared 50 mM (3.91 mg/ml) ammonium bicarbonate (AmBic) solution and 200 µl of acetonitrile (100%) into the tube. Mix well and let sit at room temperature (RT) for 5 mins.
- Remove the AmBic/acetonitrile solution and repeat the above washing step one more time.
- Dry the gel pieces in a SpeedVac, about 15 mins.
- Add 200 µl of a freshly prepared 10 mM (1.54 mg/ml) 1,4-dithiothreitol (DTT, Sigma, #10197777001) in 50 mM AmBic solution and incubate 50ºC for 30 min.
- Remove the DTT solution and wash the sample briefly with 200 µl of 50% acetonitrile. Remove the acetonitrile and dry on a SpeedVac (~15 mins).
- Add 200 µl of a freshly prepared 55 mM (10.2 mg/ml) iodoacetamide (IAA, Sigma, #I6125) in 50 mM AmBic solution and incubate at RT, in the dark, for 30 mins.
- Remove the IAA solution and wash the sample with 500 µl of 50 mM AmBic for 15 mins. Remove the AmBic and incubate with 200 µl of acetonitrile (100%). Remove the acetonitrile and dry on a SpeedVac (~15 mins).
- Incubate the dried sample in a 20 µg/ml solution of TPCK-treated trypsin (Sigma, #4352157) in 50 mM AmBic. The solution should largely cover the gel pieces. Keep in mind that the dried gel pieces will absorb the solution. Typically 500 ul of the trypsin solution are required. Incubate at 37ºC overnight.
- Terminate the reaction by incubating the sample at 100ºC for 3 mins.
- Remove the supernatant and collect in a glass clean tube. The peptides should now be in solution.
- Add 200 µl 50 mM AmBic and vortex for 15 mins. Remove and transfer the supernatant into the same glass tube.
- Add 100 µl 50 mM AmBic and 100 µl of acetonitrile (100%) and vortex for 15 mins. Remove and transfer the supernatant into the same glass tube.
- Add 200 µl acetonitrile (100%) and vortex for 15 mins. Remove and transfer the supernatant into the same glass tube.
- Repeat these 3 sequential washes one more time.
- Lyophilise the combined fractions.
- Proceed to N-glycan release, resuspend the dried material in 200 ul of 50 mM AmBic and add 1 ul of PNGaseF, incubate overnight at 37ºC.
- Condition a C18 Spe-Pak (50 mg) column (Waters, # WAT054955) with methanol (Fisher, #A452-4), 5% acetic acid, 1-propanol (Fisher, #A414-1) and 5% acetic acid. Load the PNGaseF-digested sample onto the C18 column. Collect the flow through.
- Wash the column with 4 ml of 5% acetic acid and collect the wash fractions. Pool the flow through and wash fractions, lyophilized the sample and proceed to permethylation.