Analysis of Carbohydrates on Proteins by Offline Normal-Phase Liquid Chromatography MALDI-TOF/TOF-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 141

11. Let the trypsinized solution come to room temperature.
12. Add 2.5

μ

L (2.5 U) of PNGase F and incubate for 4 h
at 37 ◦ C. Add another 2.5 L of PNGase F and continue

incubating overnight at a final enzyme concentration of 50
U/mL.
13. Stop digestion by lyophilizing samples.

μ

3.3.Purification
ofN-Glycans

This treatment removes all hydrophobic (non-polar) peptides and
enzymes from the mixture and the hydrophilic glycans will be
found in the flow-through. A protocol using classic C 18 Sep-Pak
SPE cartridges is described here, which can bind and remove
large quantities of peptides. However, for smaller total protein
amounts (up to 30

3.3.1.RPC 18 Spin
CartridgeClean-up

g), smaller spin cartridges (PepClean C 18
Spin Columns; Pierce) can be used to keep sample volumes down
(follow the manufacturer's instructions carefully).
1. After PNGase A or PNGase F digestion, redissolve the
lyophilized samples in 200

μ

L of 5% acetic acid.
2. Clamp 5-mL glass syringe onto the retort stand, attach the
C 18 Sep-Pak cartridge onto the tip and wash with 5 mL of
methanol, making sure that no air bubbles appear in the car-
tridge.
3. Wash with 5 mL of 5% acetic acid.
4. Load the sample onto the column, making sure to wash out
the sample tube.
5. Elute glycans with 3 mL of 5% acetic acid and collect this
fraction into an appropriate container as it contains free
N -glycans ( see Note 18 ).

μ

This step removes any remaining hydrophilic peptides and all salts
are converted to their corresponding acids.
1. Prepare Dowex beads by washing three times with 4 MHCl.
2. Wash repeatedly with HPLC-grade water until the pH of the
water is the same as the water supply and follow by washing
three times with 5% acetic acid ( see Note 19 ).
3. Plug a disposable polystyrene column with glass wool and
pack with 500

3.3.2.DowexCation
Exchange

L of prepared Dowex beads. Wash with two
volumes of 5% acetic acid (approx. 1 mL) ( see Note 20 ).
4. Re-suspend dry sample in 50

μ

μ L of 5% acetic acid and load

directly onto the column.
5. Elute with two volumes (approx. 1 mL) of 5% acetic acid,
collect fractions and lyophilize using a freeze dryer ( see
Note 18 ).