Rapid and High-Throughput N -Glycomic Analysis of Plant Glycoproteins - Plant Proteomics: Methods and Protocols - page 620

4. Wash twice with 200 μ L of 2 M guanidine hydrochloride,

twice with 200 μ L of water, and twice with 200 μ L of 1 %

(v/v) trimethylamine-methanol, sequentially.
5. Add 100 L of 10 % (v/v) acetic anhydride-methanol to the

well, and incubate at room temperature for 30 min.
6. Remove the solvent, and wash twice with 200

μ

μ L of 10 mM

HCl, and twice with 200 μ L of methanol.

7. Wash with 200 μ L of water.

8. Add 20 L of 2 % (v/v) acetic acid/

ACN to the well, then incubate at 80 °C for 1 h ( see Note 7 ).

μ L of aoWR solution and 180 μ

9. Elute and collect the aoWR-labeled glycans with 100 μ L of

water. Store at −20 °C ( see Note 8 ).

The HILIC treatment should be carried out just before applying
the samples to the MALDI-target plate ( see Note 9 ).

3.4 Removal of Free
Labeling Reagent by
HILIC

1. Dilute the collected glycan sample with 9 volumes of 1 % (v/v)
acetic acid/ACN.
2. A well of MassPREP HILIC 96-well Plate is washed twice
with 200

L of 1 % (v/v) acetic acid/water, and equilibrated
twice with 200

μ

L of 1 % acetic acid/95 % ACN ( see Note 4 ).
3. Load the diluted sample onto the equilibrated well of HILIC.
After naturally dropping for a few minutes, the well retaining
the glycans is washed twice with 200

μ

μ L of 1 % acetic

acid/95 % ACN.
4. Add 100 L of 1 % acetic acid/5 % ACN and collect the eluent

from the well.
5. Dry up the eluent by Speed Vac.

μ

3.5 MS Analysis of
N -Glycans

1. Dissolve the dried-up sample with 1 μ L of water, and add 1 μ L

of 10 mg/mL DHB ( see Note 10 ).
2. Spot aliquots of the sample mixture with matrix (1 L) onto

two distinct places on MTP 384 target plate ground steel, and
dry at ambient temperature.
3. The MALDI-TOF-MS spectrum is acquired in a refl ector, in
positive-ion mode, typically summing 1,000 shots on the
Burker Daltonics Autofl ex III or Ultrafl exIII ( see Note 11 ).
4. Pick possible N -glycan peaks ( m/z ) in the spectra using the
FlexAnalysis ver. 3 (Bruker Daltonics) software. The glycans'
structures are speculated using the GlycoMod Tool (http://
br.expasy.org/tools/glycomod/) and GlycoSuite Web site
(https://glycosuite.proteomesystems.com/glycosuite/gly-
codb) ( see Notes 12-14 ).
5. Normalize intensity of the isotopic peak of each glycan with
25 pmol of internal standard (GN4) for each status.

μ