May 21, 2026
Reduction and labeling of oligosaccharides/released N-glycans with Procainamide
- Kimmy Park1,2,
- Ron Orlando1,3
- 1Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602;
- 2Current address: Incyte, Wilmington, DE, 19803;
- 3GlycoScientific, Athens, GA, 30602
- Orlando Lab Protocols
Protocol Citation: Kimmy Park, Ron Orlando 2026. Reduction and labeling of oligosaccharides/released N-glycans with Procainamide. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4pm52lo5/v1
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: May 20, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 317617
Keywords: glycans with procainamide liquid chromatography, glycan derivatization, structure of glycan, labeling of oligosaccharide, glycan, procainamide liquid chromatography, oligosaccharide, released glycan, converting unreduced glcnac, other oligosaccharide, glcnac, popular tertiary amine fluorescence tag, acetylglucosamine, sugar terminus, procainamide, other reductive amination tag, reductive amination, complex chromatographic profile, ms ionization
Funders Acknowledgements:
Ron Orlando
Grant ID: R42GM113666
Abstract
Liquid Chromatography (LC)-Mass Spectrometry (MS) is a well-known tool for the characterization and quantification of N-glycans (1–5). The structure of glycans is highly fragile under the influence of MS ionization, and the unreduced N-acetylglucosamine (GlcNAc) terminus often creates complex chromatographic profiles. Derivatization of the terminus of N glycans is highly recommended in LC MS analysis. Derivatization not only significantly improves MS signal (6–9) but also avoids complex chromatographic behavior by converting unreduced GlcNAc into sugar alditols(10). Reductive amination is a common method for N-glycan derivatization (6). Procainamide (ProA) is a popular tertiary amine fluorescence tag with high ionization efficiency (8) and is a cost-effective option for research applications. This protocol describes in detail the labeling and reduction procedures for released N-glycans using ProA and sodium cyanoborohydride (NaBH₃CN), respectively. This protocol can also be used for other reductive amination tags, such as 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA), and to derivatize other oligosaccharides with a reducing-sugar terminus._
Materials
**Reagents**
- Procainamide hydrochloride (ProA - HCl) is purchased from Sigma-Aldrich (St. Louis, MO, USA), catalog# SML2088. This chemical should be stored in dark storage as it is sensitive to light.
- Acetic acid is purchased from Sigma-Aldrich (St. Louis, MO, USA), catalog# 695092.
- Dimethyl sulfoxide (DMSO) is purchased from Sigma-Aldrich (St. Louis, MO, USA), catalog# 34869.
- Sodium cyanoborohydride is purchased from Acros Organics (Branchburg, NJ, USA), catalog# AC168550500. Stored in tight container and avoid moisture.
- PD MiniTrap G10 (PDG-10) column is purchased from Cytiva (Marlborough, MA, USA), catalog# 28918010.
**Equipment**
- Pipettes and low retention tips
- 1.5 mL Eppendorf tubes
- Vortex mixer
- Incubator at 37°C
- Ventilated fume hood
- Analytical balance
- Lyophilizer or SpeedVac (Optional)
Safety warnings
Note: the safety and hazard information provided below is only a summary from the safety data sheet (SDS) published by the vendor. Please refer to the appropriate SDS for full, in-depth safety information.
**Sodium cyanoborohydride (NaBH₃CN)**
- NaBH₃CN is a flammable solid.
- NaBH₃CN releases flammable gas when in contact with water.
- NaBH₃CN can cause severe skin burns and eye damage. Fatal if swallowed, in contact with skin or if inhaled.^^11^^
**Handling and safe lab practices**
- Use NaBH₃CN only under a chemical fume hood.
- Avoid getting in the eyes, on the skin, or on clothing, and do not ingest.
- Must wear personal protective equipment/face protection, including safety goggles, protective gloves, appropriate lab coat/clothing, and approved air purifying respirator.
- Do not breathe (dust, vapor, mist, gas). Avoid dust formation.^^11^^
**Precautions for spillage or exposure**
- When a spill happens, evacuate personnel to safe areas. Use personal protective equipment as required. Ensure adequate ventilation. Keep people away from and upwind of the spill/leak. Avoid dust formation.
- If exposure happened, immediately contact medical attention and wash with water for 15 minutes if skin/eyes come in contact. 11^^
**Dimethyl sulfoxide (DMSO)**
- No hazard statement provided by the manufacturer. DMSO is identified as not a hazardous substance or mixture according to Regulation (EC) No. 1272/2008.^^12^^
**Handling and safe lab practices**
- Use appropriate safety goggles, gloves, and a lab coat when handling.
- A safety respirator is only required when vapors/aerosols are generated.
- Keep DMSO away from heat and sources of ignition.^^12^^
**Precautions for spillage or exposure**
- To clean up spillage, researchers should cover drains, collect, bind, and pump off spills.
- DMSO can have hazardous reactions when exposed to some chemicals. Refer to the safety data sheet^^12 for the full list of chemicals.
- If exposure happens, wash the contact area, including eyes or skin, with water. If swallowed, drink plenty of water and contact medical help if you feel unwell.^^12^^
**Acetic acid**
- Acetic acid is a flammable liquid and vapor.
- Acetic acid causes severe skin burns and eye damage.^^13^^
**Handling and safe lab practices**
- Keep away from open flames, hot surfaces, and sources of ignition. Take precautionary measures against static discharge.
- Keep container tightly closed in a dry and well-ventilated place.
- Use appropriate safety goggles, gloves, and lab coat when handling.
- A safety respirator is only required when vapors/aerosols are generated.^^13^^
**Precautions for spillage or exposure**
- When spillage happens, cover drains, collect, bind, and pump off spills. Observe possible ignition sources or reactive chemicals.^^13 Clean up the spillage with a liquid-absorbent and neutralizing material.
- If exposure happened, wash the contact area, including eyes or skin, with water. Remove to fresh air if inhaled. Contact medical help immediately.
- If swallowed, do not induce vomiting, rinse mouth with water, and contact medical help if you feel unwell.^^13^^
Reagents Preparation
Prepare a labeling solution.
Mix a 7:3 (DMSO/acetic acid) solution. For example, 1 mL of labeling solution needs 700 µL DMSO and 300 µL Acetic acid.
For every 1mL of labeling solution:
Weight 108 mg of Procainamide – HCl if the analyte of interest is released N-glycans from an antibody.
Weight 216 mg of Procainamide-HCl if the analyte of interest is oligosaccharides. Optional: this ratio can also be used to label released N-glycans from cell pellets/antibody.
Add the labeling solution to the weighted Procainamide - HCl. Vortex until fully dissolved.
For every 1mL of labeling solution:
Weight 63 mg of NaBH₃CN if the analyte of interest is released N-glycans from the antibody.
Weight 126 mg of NaBH₃CN if the analyte of interest is oligosaccharides. Optional: This ratio can also be used to label released N-glycans from cell pellets/antibody.
Mix ProA-HCl into the labeling solution with weighted NaBH₃CN. Vortex until fully dissolved.
Note: NaBH₃CN can appear not to solubilize well. Approximately 20 µL of water can be slowly added for every 6 mg of NaBH₃CN to aid solubilization. This procedure must be performed only in a ventilated fume hood.
Protocol
Dissolve 1 mg of oligosaccharides or N-glycans released from 1 mg of glycoprotein with 10 µL of water.
Add 200 µL of the labeling solution to the solubilized oligosaccharides/released N-glycans. The mixture is incubated at 37°C overnight.
After incubation, 100 µL of acetone is added to quench the reaction at 65°C for 30 minutes.
Note: Every 5 minutes, open the lid and sonicate for 10 seconds to release gas. Repeat this step 3 - 4 times.
The excess labeling reagents are cleaned up using a size-exclusion PD MiniTrap G10 column:
Add 140-240 µL 5% acetic acid to each sample (max PDG10 loading volume is 300 µL)
Shake/vortex PDG-10 column to resuspend the stationary phase, then allow to settle while the column is vertical.
Drain storage solution out of column
Flush column with 2 mL 5% acetic acid (x3)
Load sample and allow sample to enter the stationary phase fully
Add 5% acetic acid so that (sample volume + this volume) = 0.7 mL. For example, if the sample is 140 µL, add 560 µL 5% acetic acid
Allow the mobile phase to enter the bed fully.
Add 600 µL 5% acetic acid, collect eluent.
The collected fractions can be dried down via lyophilizer/SpeedVac prior to analysis or storage at -80°C. Although not evaluated, the lyophilized labeled N-glycans or oligosaccharides can stay stable at -80°C for approximately 6 months.
Protocol references
(1) Zhou, S.; Hu, Y.; Veillon, L.; Snovida, S. I.; Rogers, J. C.; Saba, J.; Mechref, Y. Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT. Anal Chem 2016, 88 (15), 7515-7522. DOI: 10.1021/acs.analchem.6b00465.
(2) Zhou, S.; Hu, Y.; DeSantos-Garcia, J. L.; Mechref, Y. Quantitation of permethylated N-glycans through multiple-reaction monitoring (MRM) LC-MS/MS. J Am Soc Mass Spectrom 2015, 26 (4), 596-603. DOI: 10.1007/s13361-014-1054-1.
(3) Suzuki, N.; Abe, T.; Natsuka, S. Quantitative LC-MS and MS/MS analysis of sialylated glycans modified by linkage-specific alkylamidation. Anal Biochem 2019, 567**, 117-127. DOI: 10.1016/j.ab.2018.11.014 .
(4) Ruhaak, L. R.; Miyamoto, S.; Lebrilla, C. B. Developments in the identification of glycan biomarkers for the detection of cancer. Mol Cell Proteomics 2013, 12 (4), 846-855. DOI: 10.1074/mcp.R112.026799.
(5) Jin, M.; Kim, J.; Ha, J.; Kim, A.; Lee, J.; Park, C. S.; Kang, M.; Kim, J.; Mun, C.; Kim, J.; et al. Identification and quantification of sialylated and core-fucosylated N-glycans in human transferrin by UPLC and LC-MS/MS. Anal Biochem 2022, 647**, 114650. DOI: 10.1016/j.ab.2022.114650.
(6) Ruhaak, L. R.; Zauner, G.; Huhn, C.; Bruggink, C.; Deelder, A. M.; Wuhrer, M. Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem 2010, 397 (8), 3457-3481. DOI: 10.1007/s00216-010-3532-z .
(7) Lauber, M. A.; Yu, Y. Q.; Brousmiche, D. W.; Hua, Z.; Koza, S. M.; Magnelli, P.; Guthrie, E.; Taron, C. H.; Fountain, K. J. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal Chem 2015, 87 (10), 5401-5409. DOI: 10.1021/acs.analchem.5b00758.
(8) Zhou, S.; Veillon, L.; Dong, X.; Huang, Y.; Mechref, Y. Direct comparison of derivatization strategies for LC-MS/MS analysis of N-glycans. Analyst 2017, 142 (23), 4446-4455. DOI: 10.1039/c7an01262d .
(9) Helali, Y.; Delporte, C. Updates of the current strategies of labeling for N-glycan analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2024, 1237**, 124068. DOI: 10.1016/j.jchromb.2024.124068 .
(10) Jensen, P. H.; Karlsson, N. G.; Kolarich, D.; Packer, N. H. Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc 2012, 7 (7), 1299-1310. DOI: 10.1038/nprot.2012.063.
(11) Safety Data Sheet: Sodium cyanoborohydride; ThermoFisher Scientific, 2025. (accessed 05/13/2026).
(12) Safety Data Sheet: Dimethyl sulfoxide Sigma-Aldrich, 2026. (accessed 05/13/2026).
(13) Safety Data Sheet: Acetic acid; Sigma-Aldrich, (accessed 05/17/2026).