Waters GlycoWorks High-throughput Sample Preparation Kit - Manual (Page 3)

GlycoWorks High-throughput Sample Preparation Kit 3
[ CARE AND USE MANUAL ]
Step 1. Denaturation and Enzymatic Deglycosylation
RapiGest, DTT, IAM are included in the GlycoWorks Reagent Kit box labeled
“Enzymatic Deglycosylation Reagents” for your convenience in following
this protocol:
1) Prepare RapiGest solution by adding 500 μL of 25 mM ammonium
bicarbonate to 1 mg RapiGest vial.
2) For the GlycoWorks Control Standard, add 100 μL of 25 mM
ammonium bicarbonate to vial.
3) Add 90 µL of the 0.2% RapiGest solution to the glycoprotein
solution in a sterile 1.5 mL centrifuge tube.
Note: For use with other samples, it is recommended that the protein
concentration be 1–5 mg/ml.
3) Prepare 0.5 M DTT by adding 500 µL of 25 mM ammonium bicarbon-
ate to the tube. Vortex to mix into solution.
■
Add 2 μL of 0.5 M DTT and incubate sample(s) at 37 °C for
30 minutes. Cool to room temperature.
4) Prepare 0.5 M IAM by adding 500 µL of 25 mM ammonium
bicarbonate to the tube. Vortex to mix into solution.
■
Iodoacetamide is light sensitive. Cover the tube in foil and
place it in a dark drawer when not in use. This solution should
be made fresh daily.
■
Add 4 μL of 0.5 M IAM and incubate at room temperature in
the dark for 30 minutes.
5) Add 2.5 mU (1 μL of 2.5 mU μL-1 stock) of PNGase F to each
sample and incubate at 37 °C overnight. If a more concentrated
enzyme stock is purchased, dilute with included buffer first to get
to a 2.5 mU/µl concentration.
Note: If digesting glycoprotein samples greater than 1 mg/mL, it is
recommended to use more PNGase F.
Step 2a. Extracting the Released Glycans
Note: There may be variation in flow rates from well to well when using
this plate with the vacuum manifold. General guidelines are provided to
help minimize this. However, settings will depend on the specific vacuum
and will need to be optimized. If well to well reproducibility is critical,
using a positive pressure manifold produces consistent reproducibility.
1) Condition the GlycoWorks HILIC μElution plate by adding 200 μL of
Milli-Q ® water to each well and aspirate using the vacuum manifold
or positive pressure manifold.
2) Add 200 μL of 85% acetonitrile to each well and aspirate using the
vacuum manifold or positive pressure manifold.
3) Take 50 μL of the PNGase F-digested protein mix and add this to
350 μL of acetonitrile.
Note: If precipitation occurs, do not centrifuge! Centrifugation causes
reduced glycan recovery.
4) Load the sample onto the resin bed using a very low vacuum setting
(1–2 in Hg) or positive pressure manifold.
5) Wash each well 3 times with 200 µL of 85% acetonitrile, pooling
the filtrate after each wash into the waste tray for disposal.
6) Remove the waste tray and replace with a 96-well collection plate.
7) Elute the glycans with 50 μL of 100 mM ammonium acetate in 5%
acetonitrile.
Note: The concentration of acetonitrile and ammonium acetate in the
elution buffer may require optimization for some glycan samples.
8) Repeat the elution two more times.
9) Place the collection plate and dry released glycans by vacuum
evaporation to a concentrate of ≤ 1 µL in volume.
Note: If a rotor is not available for the vacuum centrifugal evaporator,
transfer each collected elution into a sterile 1.5 (or 0.5) mL Eppendorf
and dry down by vacuum evaporation.
Step 1 Tips and Tricks
■
The minimum amount of glycoprotein advised is 10 μg
(100 μL of 0.1 mg/mL).
■ Avoid temperature extremes with glycan samples.
■
Exposure to high pH may result in epimerization of
reducing sugars.