Nov 27, 2025
Preparation method of pyridylaminated N-glycans
- 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
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 29, 2025
Last Modified: November 27, 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
BlotGlycoSumitomo BakeliteCatalog #BS-45414
Rapid PNGase FNew England BiolabsCatalog #P0710S
Sample preparation 1: for glycoprotein
1m
50-100 µg glycoprotein (dry sample) in 0.2 mL PCR tube
add 15 µL of DDW
30s
vortex
30s
Sample preparation 2: for human serum
1m
1 µL of human serum in 0.2 mL PCR tube
add 15 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 16 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
(10 µL of 10% NP-40, 990 µL of DDW)
1m
vortex
30s
transfer 16 µL (protein content 50-100 µg) to 0.2 mL PCR tube
30s
Enzymatic dygestion using Rapid PNGase F
13m 30s
add 4 µL of Rapid PNGase F Buffer (5x) to sample solution, mix gently with a pipette—total reaction volume to 20 µL
Rapid PNGase FNew England BiolabsCatalog #P0710S
30s
denature at 80 °C for 00:02:00
2m
cool down the sample solution
30s
add 1 µL of Rapid PNGase F, mix by pipetting
30s
incubate at 50 °C for00:10:00
10m
Glycan purification and fluorescent labeling using BlotGlyco
8h 59m 10s
50 µL of polymer beads suspension in the reaction tube
BlotGlycoSumitomo BakeliteCatalog #BS-45414
30s
wash polymer beads with 100 µL of DDW, twice, spin down to remove water completely
Note
Modification from manufacturer's protocol: wash polymer beads with 100 µL of DDW, twice
1m
replace the reaction tube into a new 1.5 mL tube
10s
add 180 µL of 2% acetic acid in acetonitrile to the reaction tub20 µL e
(20 µL of980 µL 980 µL a980 µL c980 µL etic 980 µL aci980 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 80 °C for 01:00:00 , without cover (the solvent has completely evaporated.)
(If the beads are not dry completely, continue heating for another 00:15:00 )
1h
replace the reaction tube into the 2 mL tube
30s
wash with 200 µL of 2M guanidine solution
(1.9 g guanidine hydrochloride, 10 mL of DDW)
1m
wash with 200 µL of DDW, mix by pipetting
Note
Modification from manufacturer's protocol: mix by pipetting.
1m
wash with 200 µL of DDW
1m
wash with 200 µL of 1% triethylamine in MeOH, mix by pipetting
(100 µL of triethylamine, 9.9 mL of MeOH)
Note
Modification from manufacturer's protocol: mix by pipetting.
1m
wash with 200 µL of 1% triethylamine in MeOH
1m
replace the reaction tube into the 1.5 mL tube
10s
add 100 µL of 1% triethylamine in MeOH, mix by pipetting
(15 µL of acetic anhydride, 135 µL of MeOH)
Note
Modification from manufacturer's protocol: mix by pipetting.
1m
leave at room temperature for 00:30:00
30m
replace the reaction tube into the 2 mL tube
10s
spin down to remove the fluid
30s
wash with 200 µL of DDW, twice
2m
replace the reaction tube into the 1.5 mL tube
10s
add 20 µL of DDW
30s
add 180 µL of 2% acetic acid in acetonitrile, mix by pipetting
(100 µL of acetic acid, 4.9 mL of acetonitrile)
Note
Modification from manufacturer's protocol: mix by pipetting.
1m
heat at 70 °C for 01:30:00 , without cover (the solvent has completely evaporated.)
(If the beads are not dry completely, continue heating for another 00:15:00
1h 30m
add 30 µL PAtion reagent
(138 mg of 2-aminopyridine, 50 µL of acetic acid)
30s
heat at 80 °C for 01:00:00
1h
add 110 µL of reduction reagent , mix by pipetting
(100 mg of Me2NH-borane, 40 µL of acetic acidtic acid, 25 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
13.200 rpm, 25°C, 00:01:00
1m 30s
add 1080 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 200 µL of DDW
1m
washed with 200 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
2000 rpm, 25°C, 00:01:00 and 13200 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 600 µL of 95%(v/v) acetonitrile, cfg. 2000 rpm, 25°C, 00:01:00 and 13200 rpm, 25°C, 00:00:30
(1.9 mL of CH3CN, 100 µL of DDW)
2m
wash with 600 µL of 95%(v/v) acetonitrile, 2000 rpm, 25°C, 00:01:00 and 13200 rpm, 25°C, 00:00:30
2m
replace the reaction tube into a new 1.5 mL tube
10s
add 25 µL of DDW
30s
2000 rpm, 25°C, 00:01:00 and 13200 rpm, 25°C, 00:00:30
add 25 µL of 0.1% acetic acid
(1% acetic acid: 10 µL of 100% acetic acid, 990 µL of DDW)
(0.1% acetic acid: 10 µL of 1% acetic acid, 90 µL of DDW)
30s
2000 rpm, 25°C, 00:01:00 and 13200 rpm, 25°C, 00:00:30
2m
transfer collected solution to 0.6 mL PCR tube with 200 µL of 0.28% ammonium aq.
(2.8% ammonium aq.: 200 µL of 28% ammonium aq., 1.8 mL of DDW)
(0.28% ammonium aq.: 100 µL of 2.8% ammonium aq., 900 µL of DDW)
Note
A step to remove O-acetylated by-products
2m
heat at 70 °C for 00:30:00
30m
lyophilize
3h
dissolved in 100 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