Biology Reference
In-Depth Information
4. Add 1.6 mL 100 mM ammonium bicarbonate to achieve a
fi nal concentration of 0.99 M urea.
5. Add trypsin to achieve a fi nal trypsin-glycoprotein sample ratio
of 1:20 (5
μ
g trypsin-100
μ
g glycoprotein) and incubate for
16 h at 37 °C
Equilibration step: Pass the following solutions sequentially through
the PGC column and discard the fl ow through:
3.7 Affi nity
Purifi cation of
N-Glycopeptides Using
a Porous Graphitic
Carbon (PGC) Column
1. 1 mL 1 M NaOH.
2. 2 mL water.
3. 1 mL 30 % acetic acid.
4. 2 mL water.
5. 1 mL elution solvent (50 % acetonitrile, 0.1 % formic acid
[v/v] in water).
6. 1 mL wash solvent (5 % acetonitrile, 0.1 % formic acid [v/v] in
water).
Do not allow air to enter the column.
Loading step:
1. Adjust the pH of the samples to 5.0 with 0.1 % trifl uoroacetic
acid (TFA).
2. Slowly load the sample (approximately 1 drop/s) onto the column
followed by 1 mL water. Collect the fl ow through for subsequent
analysis by MS if needed to evaluate the unbound peptides.
Desalting step:
1. Pass 1 mL wash solvent through the column and recover the
fl ow through for subsequent analysis by MS if needed. This
sample can then be desalted using a conventional reverse phase
C18 solid phase extraction prior to analysis by MS (
see
Note 5
).
Elution step:
1. Pass 1 mL elution solvent through the cartridge bed and collect
the fl ow through.
2. Gently pass air through the column to elute all the solvent into
a collection tube.
3. Dry the sample in a rotary evaporator (e.g., Savant) prior to
downstream MS analysis.
After the glycoprotein digestion a pool of peptides and glyco-
peptides will be present in the enriched samples. After the desalting
and cleanup steps various downstream experiments are possible.
These include protein sequence identifi cation, characterization of
glycopeptide sequences, determination of the glycosylation sites
and interpretation of
N
-glycan structure in the
N
-glycopeptides.
