Nov 28, 2025

Public workspacePreparation method of pyridylaminated N-glycans

PLOS One
DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjez1pgk5/v1
  • Riko Makino1,
  • Shunji Natsuka1
  • 1Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University 8050 Ikarashi-nino-cho, Nishi-ku, Niigata 950-2181, Japan
  • Riko Makino: https://researchmap.jp/Riko_Makino
  • Shunji Natsuka: https://researchmap.jp/read0014427
理子 牧野
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjez1pgk5/v1
External link: https://doi.org/10.1371/journal.pone.0336565
Protocol Citation: Riko Makino, Shunji Natsuka 2025. Preparation method of pyridylaminated N-glycans. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjez1pgk5/v1
Manuscript citation:
Makino R, Natsuka S (2025) Optimizing the preparation of labeled N-glycans for rapid, simplified, and high-precision analysis. PLOS One 20(12). doi: 10.1371/journal.pone.0336565
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 30, 2025
Last Modified: November 28, 2025
Protocol Integer ID: 228515
Keywords: N-glycan, human serum, human urine, cultured cells, glycan, glycans this protocol, preparation of human serum, preparation method, purification, blotglyco, aminopyridine, protein sample, fluorescent labeling, preparation
Abstract
This protocol describes the purification of N-glycans from approximately 50 µg of protein samples, followed by fluorescent labeling with 2-aminopyridine. The procedure utilizes Rapid PNGase F (New England BioLabs) and BlotGlyco (Sumitomo Bakelite). Representative examples include the preparation of human serum, human urine, and CHO-K1 membrane fractions; however, the protocol is also applicable to other types of samples.
Protocol materials
ReagentBlotGlycoSumitomo BakeliteCatalog #BS-45414
ReagentRapid PNGase FNew England BiolabsCatalog #P0710S
Troubleshooting
Sample preparation 1: for glycoprotein
1m
50-100 µg glycoprotein (dry sample) in 0.2 mL PCR tube
add Amount15 µL of DDW
30s
vortex
30s
Sample preparation 2: for human serum
1m
Amount1 µL of human serum in 0.2 mL PCR tube
add Amount15 µL of DDW
30s
vortex
30s
Sample preparation 3: for human urinary protein
1h
50 µg of human urinary protein ppt. (dry sample) in 0.2 mL PCR tube
add Amount16 µL of DDW
30m
vortex
30m
Sample preparation 4: for CHO-K1 cell membrane fraction
2m
1 mg of CHO-K1 cell membrane fraction (dry sample) in 0.2 mL PCR tube
add XX µL of 0.1% NP-40
(Amount10 µL of 10% NP-40, Amount990 µL of DDW)
1m
vortex
30s
transfer Amount16 µL (protein content 50-100 µg) to 0.2 mL PCR tube
30s
Enzymatic dygestion using Rapid PNGase F
13m 30s
add Amount4 µL of Rapid PNGase F Buffer (5x) to sample solution, mix gently with a pipette—total reaction volume to Amount20 µL
ReagentRapid PNGase FNew England BiolabsCatalog #P0710S
30s
denature at Temperature80 °C for Duration00:02:00
2m
cool down the sample solution
30s
add Amount1 µL of Rapid PNGase F, mix by pipetting
30s
incubate at Temperature50 °C forDuration00:10:00
10m
Glycan purification and fluorescent labeling using BlotGlyco
8h 59m 10s
Amount50 µL of polymer beads suspension in the reaction tube
ReagentBlotGlycoSumitomo BakeliteCatalog #BS-45414

30s
wash polymer beads with Amount100 µL of DDW, twice, spin down to remove water completely
Note
Modification from manufacturer's protocol: wash polymer beads with Amount100 µL of DDW, twice


1m
replace the reaction tube into a new 1.5 mL tube
10s
add Amount180 µL of 2% acetic acid in acetonitrile to the reaction tubAmount20 µL e
(Amount20 µL ofAmount980 µL Amount980 µL aAmount980 µL cAmount980 µL etic Amount980 µL aciAmount980 µL d of acetonitrile)
1m
add sample solution to the reaction tube, mix by pipetting
Note
Modification from manufacturer's protocol: mix by pipetting.

30s
heat at Temperature80 °C for Duration01:00:00 , without cover (the solvent has completely evaporated.)
(If the beads are not dry completely, continue heating for another Duration00:15:00 )
1h
replace the reaction tube into the 2 mL tube
30s
wash with Amount200 µL of 2M guanidine solution
(Amount1.9 g guanidine hydrochloride, Amount10 mL of DDW)
1m
wash with Amount200 µL of DDW, mix by pipetting
Note
Modification from manufacturer's protocol: mix by pipetting.

1m
wash with Amount200 µL of DDW
1m
wash with Amount200 µL of 1% triethylamine in MeOH, mix by pipetting
(Amount100 µL of triethylamine, Amount9.9 mL of MeOH)
Note
Modification from manufacturer's protocol: mix by pipetting.

1m
wash with Amount200 µL of 1% triethylamine in MeOH
1m
replace the reaction tube into the 1.5 mL tube
10s
add Amount100 µL of 1% triethylamine in MeOH, mix by pipetting
(Amount15 µL of acetic anhydride, Amount135 µL of MeOH)
Note
Modification from manufacturer's protocol: mix by pipetting.

1m
leave at room temperature for Duration00:30:00
30m
replace the reaction tube into the 2 mL tube
10s
spin down to remove the fluid
30s
wash with Amount200 µL of DDW, twice
2m
replace the reaction tube into the 1.5 mL tube
10s
add Amount20 µL of DDW
30s
add Amount180 µL of 2% acetic acid in acetonitrile, mix by pipetting
(Amount100 µL of acetic acid, Amount4.9 mL of acetonitrile)
Note
Modification from manufacturer's protocol: mix by pipetting.

1m
heat at Temperature70 °C for Duration01:30:00 , without cover (the solvent has completely evaporated.)
(If the beads are not dry completely, continue heating for another Duration00:15:00
1h 30m
add Amount30 µL PAtion reagent
(Amount138 mg of 2-aminopyridine, Amount50 µL of acetic acid)
30s
heat at Temperature80 °C for Duration01:00:00
1h
add Amount110 µL of reduction reagent , mix by pipetting
(Amount100 mg of Me2NH-borane, Amount40 µL of acetic acidtic acid, Amount25 µL of DDW)
Note
Modification from manufacturer's protocol: mix by pipetting.

30s
heat at 80°C for 35 min
35m
replace the reaction tube into a new 1.5 mL tube
10s
Centrifigation13.200 rpm, 25°C, 00:01:00

1m 30s
add Amount1080 µL of acetonitrile to prepare 90% acetonitrile sample aliquot
Note
Modification from manufacturer's protocol: prepare 90% acetonitrile sample aliquot, considering the volatility of acetic acid.

1m
add the sample aliquot to the cleanup column in a centrifuge
10s
washed with Amount200 µL of DDW
1m
washed with Amount200 µL of acetonitrile, twice
2m
wlet the fluid drop until completely loaded

Note
Modification from manufacturer's protocol: wait until all the liquid has been loaded onto the column.

20m
Centrifigation2000 rpm, 25°C, 00:01:00 and Centrifigation13200 rpm, 25°C, 00:00:30
Note
Modification from manufacturer's protocol: apply the solution to the column by gentle centrifugation, then spin briefly at high speed to pass the liquid through. Perform the following centrifugation steps in the same manner.

2m
wash with Amount600 µL of 95%(v/v) acetonitrile, cfg. Centrifigation2000 rpm, 25°C, 00:01:00 and Centrifigation13200 rpm, 25°C, 00:00:30
(Amount1.9 mL of CH3CN, Amount100 µL of DDW)
2m
wash with Amount600 µL of 95%(v/v) acetonitrile, Centrifigation2000 rpm, 25°C, 00:01:00 and Centrifigation13200 rpm, 25°C, 00:00:30
2m
replace the reaction tube into a new 1.5 mL tube
10s
add Amount25 µL of DDW
30s
Centrifigation2000 rpm, 25°C, 00:01:00 and Centrifigation13200 rpm, 25°C, 00:00:30
add Amount25 µL of 0.1% acetic acid
(1% acetic acid: Amount10 µL of 100% acetic acid, Amount990 µL of DDW)
(0.1% acetic acid: Amount10 µL of 1% acetic acid, Amount90 µL of DDW)
30s
Centrifigation2000 rpm, 25°C, 00:01:00 and Centrifigation13200 rpm, 25°C, 00:00:30
2m
transfer collected solution to 0.6 mL PCR tube with Amount200 µL of 0.28% ammonium aq.
(2.8% ammonium aq.: Amount200 µL of 28% ammonium aq., Amount1.8 mL of DDW)
(0.28% ammonium aq.: Amount100 µL of 2.8% ammonium aq., Amount900 µL of DDW)
Note
A step to remove O-acetylated by-products

2m
heat at Temperature70 °C for Duration00:30:00
30m
lyophilize
3h
dissolved in Amount100 µL of DDW
1m
RP-LC/MS analysis

Note
LC/MS condition:
Hanzawa K, Suzuki N, Natsuka S. Structures and developmental alterations of N-glycans of zebrafish embryos. Glycobiology, 2017;27(3):228–245. DOI: 10.1093/glycob/cww124