Biology Reference
In-Depth Information
1. MultiScreen Solvent fi lter plate (Millipore, Billerica, USA) and
vacuum manifold (Waters, Milford, USA).
2. Oligosaccharide Purifi cation Kit BlotGlyco ® (SUMITOMO
BAKELITE, Tokyo, Japan): BlotGlyco ® beads (Hydrazide
Functionalized Polymer) [ 18 ], N
2.3 Glycoblotting
-((aminooxy)acetyl)trypto-
phanylarginine methyl ester (aoWR) [ 17 ].
3. 50
α
M chitotetraose (GN4): Dissolve 50 mg of GN4 in
1.2 mL of water to prepare a 50 mM solution. Store at −20 °C.
Dilute to 50
μ
M before use.
4. 2 % (v/v) acetic acid/ACN: Mix 9.8 mL of ACN and 0.2 mL
of acetic acid. Store at room temperature.
5. 2 M guanidine hydrochloride: Dissolve 1.9 g of guanidine
hydrochloride in 10 mL of water. Store at room temperature.
6. 1 % (v/v) trimethylamine-methanol: Mix 9.9 mL of trimethyl-
amine and 0.1 mL of methanol. Store at room temperature.
7. 10 % (v/v) acetic anhydride-methanol: Mix 100
μ
μ
L of acetic
L of methanol. Prepare before use.
8. 10 mM HCl: Store at room temperature.
anhydride and 900
μ
2.4 Removal of Free
Labeling Reagent by
Hydrophilic Interaction
Chromatography
(HILIC)
1. MassPREP HILIC 96-well Plate (Waters, Milford, USA).
2. 1 % acetic acid/99 % ACN: Mix 9.9 mL of ACN and 0.1 mL
of acetic acid.
3. 1 % acetic acid/95 % ACN: Mix 9.5 mL of ACN, 0.1 mL of
acetic acid and 0.4 mL of water.
4. 1 % acetic acid/5 % ACN: Mix 0.5 mL of ACN, 0.1 mL acetic
acid and 9.4 mL of water.
5. 1 % (v/v) acetic acid/water: Mix 0.1 mL of acetic acid and
9.9 mL of water.
1. 10 mg/mL 2,5-dihydroxybenzoic acid (DHB): Dissolve
10 mg of DHB in 1 mL of 30 % ACN.
2. MTP 384 target plate ground steel TF (Burker Daltonics,
Billerica, USA).
2.5 MS Analysis of
N -Glycans
3
Methods
1. Freeze-dry 50
g of glycoproteins ( see Note 1 ).
2. Dissolve the glycoprotein sample in 20
μ
3.1 Sample
Preparation
L of 50 mM citrate-
phosphate buffer (pH 5.0) and incubate at 60 °C for 10 min.
3. Add 2
μ
L of 100 mM DTT to the sample and incubate at
60 °C for 30 min. After adding 3
μ
L of 100 mM iodoacet-
amide, the mixture is incubated in the dark at room tempera-
ture for 30 min ( see Note 2 ).
μ
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