Biomedical Engineering Reference
In-Depth Information
L of 100% acetonitrile and apply to the cartridge
allowing time for it to penetrate into the membrane.
5. Wash the cartridge with 1 mL of 100% acetonitrile and allow
to drain.
6. Wash five times with 1 mL 96% acetonitrile solution, allow-
ing each aliquot to drain before the next step is applied. Dis-
card the flow-through.
7. Place the cartridge over a collection vessel suitable for drying
1.5 mL of water and elute glycans with three washes of 0.5
mL water, allowing each wash to drain before the next one
is applied.
8. Lyophilize samples (
see
Note 24
).
with 100
μ
1. Dissolve
N
-glycan oligosaccharides in 80% acetonitrile and
load onto an amide-80 column (300
3.7.Normal-Phase
Chromatography
μ
m
×
25 cm;
5
m bead size; LC Packings/Dionex) and with the cap-
illary HPLC system, generate a gradient that flows at
3
μ
L/min.
2. Elute oligosaccharides with increasing aqueous concentra-
tion: equilibrate the NP column with 5% A. For elution of
N
-glycans, the gradient is initiated 5 min after injection and
increased linearly to 52% A over 96 min. The column is then
equilibrated for 10 min with 5% solvent A prior to the next
injection.
3. If using a UV detector, monitor at 254 nm for 2-AA- and
2-AB-labelled glycans.
4. Pass column effluent directly to a Probot sample fraction-
ation system and spot onto a MALDI target plate at 20 s
intervals. Leave spots to air-dry.
μ
3.8.Acquisition
ofMALDI-TOF-MS
1. Overlay all sample spots with 0.6
L of 2,5 DHB solu-
tion (10 mg/mL in 50% methanol) and rapidly dry
in a vacuum desiccator. This drying method produces
small crystals which facilitate easy spectral acquisition (
see
Note 25
).
2. Fire the laser on a few spots over the plate in order to deter-
mine the correct laser intensity for the automatic acquisition
of the MS data.
3. Acquire spectra automatically by exposing each spot to
1,500-2,500 shots over a range between
m/z
850 and
5,000.
μ
3.9.AnalysisofMS
Results
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