Biology Reference
In-Depth Information
4. Up to 96 samples can be treated simultaneously by using a
MultiScreen Solvent fi lter plate and a MassPREP HILIC
96-well Plate.
5. The sample glycans and internal standards should be blended
in a balanced manner. It is not recommended to use the inter-
nal standards for analyzing the sample without estimating the
glycan contents.
6. Dry up until the smell of acetic acid completely disappears.
7. The glycoblotting method can be applied to reductive amination
with common fl uorescent dyes such as 2-aminobenzamide [
20
].
8. The sample should be subjected to the HILIC treatment
within a week.
9. When the surplus labeling reagent is removed from the sample
mixture, the label compound bound to the glycan will be
released through chemical disequilibrium.
10. The laser shots should hit the crystals on the periphery of the
sample spot with DHM matrix, in order to acquire stable mass
spectra.
11. The number, power and gain of the laser shots should be con-
stant for quantitative analysis. Stable isotope-coded derivatiza-
tion is also available and useful [
16
-
18
].
12. Difference (
std
) from the theoretical mass value of an internal
standard (M+H 831.33533
m/z
) was determined in each
measurement. The identifi cation of glycan was manually per-
formed with the following parameters:
Δ
Δ
value of glycan
std
− 0.3 Da.
13. Acidic sugars such as
N
-acethylneuramic acid, sialic acid, gluc-
uronic acid, sulfate and phosphate-contained saccharides have
never been detected in Plant
N
-glycans.
14. It should be noted that artifi cially methylated glycans (+14
m/z
) could be detected in the MS spectrum.
<2 Da,
Δ
std
+ 0.3 Da >
Δ
>
Δ
Acknowledgments
This research was supported by Grants-in-Aid for Scientifi c Research
(B) (22380186) and Scientifi c Research on Innovative Areas
(22114507) from the Ministry of Education, Culture, Sports, Science,
and Technology, Japan, to TM. KK was a holder of a Research
Fellowship of the Japan Society for the Promotion of Science.
