Waters GlycoWorks Single Use 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
1) Condition each cartridge by adding 1,000 µL of Milli-Q ® water to
each cartridge and aspirate using the vacuum manifold.
2) Add 1,000 µL of 85% acetonitrile to each cartridge and aspirate
using the vacuum manifold.
3) Take 100 µL of the PNGase F-digested protein mix and add this to
700 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).
5) Wash each cartridge 3 times with 400 µL of 85% acetonitrile,
collecting the filtrate after each wash into the waste tray.
6) Remove the waste tray and replace with sterile 1.5 mL Eppendorf
tubes.
7) Elute the glycans with 100 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 ≤ 2 µL in volume.
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.