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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

Abstract

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Introduction

N-glycosylation of human proteins is a common and essential post-translational modification1 and may help predict the occurrence and development of diseases relatively accurately. Due to the complexity of its structure, it is expected that there are more than 5,000 glycan structures, providing great potential as diagnostic and predictive biomarkers for diseases2. N-glycans attached to immunoglobulin G (IgG) have been shown to be essential for IgG's function, and IgG N-glycosylation participates in the balance between the pro- and anti-inflammatory systems3. Different....

Protocol

All the subjects included in the protocol have been approved by the Ethics Committee of the Capital Medical University, Beijing, China12. Written informed consent was obtained from each subject at the beginning of the study.

1. IgG isolation

  1. Prepare the chemicals including binding buffer (phosphate buffered saline, PBS): 1x PBS (pH = 7.4), neutralizing buffer: 10x PBS (pH = 6.6-6.8), eluent: 0.1 M formic acid (pH = 2.5), neutralizing solution for eluent: 1 M ammonium bicarbonate, stored buffer: 20% ethanol + 20 mM Tris + 0.1 M NaCl (pH = 7.4), cleaning solution for protein G: 0.1 M NaOH + 30% propan-2-ol.....

Representative Results

As shown in Figure 1, IgG N-glycans were analyzed into 24 initial IgG glycan peaks (GPs) based on peak position and retention time. The N-glycan structures are available through mass spectrometry detection according to a previous study (Table 1)15. To ensure that the results were comparable, total area normalization was applied, in which the amount of glycans in each peak was expressed as a percentage.......

Discussion

UPLC serves as a relative quantitative analysis method5,15. The results indicate that UPLC is a stable detection technology with good reproducibility and relative quantitative accuracy. The amount of glycans in each peak is expressed as a percentage of the total integrated area using UPLC, which is the relative value. The relative quantification improves the comparability of test samples. In addition, 96 well protein G plates are used to purify IgG with 96 sample.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (81673247 & 81872682) and Australian-China Collaborative Grant (NH & MRC - APP1112767 -NSFC 81561128020).

....

Materials

NameCompanyCatalog NumberComments
2-aminobenzamide, 2-ABSigma, China
96-well collection plateAXYGEN
96-well filter platePol0.45 um GHP
96-well monolithic plateBIA Separations
96-well plate rotorEppendorf Co., Ltd, GermanyT_1087461900
Acetic acidSigma, China
AcetonitrileHuihai Keyi Technology Co., Ltd, China
Ammonium bicarbonateShenggong Biological Engineering Co., Ltd, China
Ammonium formateBeijing Minruida Technology Co., Ltd.
Constant shaking incubator/rockerZhicheng analytical instrument manufacturing co., Ltd, ChinaZWY-10313
Dextran Calibration Ladder/Glycopeptide columnWatts technology Co., Ltd, ChinaBEH column
Dimethyl sulfoxide (DMSO)Sigma, China
Disodium phosphateShenggong Biological Engineering Co., Ltd, China
Electric ovensTester instruments Co., Ltd202-2AB
Empower 3.0Waters technology Co., Ltd, America
EthanolHuihai Keyi Technology Co., Ltd, China
Formic acidSigma, China
GlycoProfile 2-AB Labeling kitSigma, China
HClJunrui Biotechnology Co., Ltd, China
High-speed centrifugeEppendorf Co., Ltd, Germany5430
IgepalSigma, China
Low temperature centrifugeEppendorf Co., Ltd, Germany
Low temperature refrigeratorQingdao Haier Co., Ltd
Manifold 96-well plateWatts technology Co., Ltd, China186001831
MethanolHuihai Keyi Technology Co., Ltd, China
Milli-Q pure water meterMillipore Co., Ltd, AmericaAdvantage A10
NaOHShenggong Biological Engineering Co., Ltd, China
PH testerSartorius Co., Ltd, GermanyPB-10
Phosphate buffered saline, PBSShenggong Biological Engineering Co., Ltd, China
PipetteEppendorf Co., Ltd, Germany4672100, 0.5-10μl & 10-100μl & 20-200μl & 1000μl
PNGase F enzymeSigma, China
Potassium dihydrogen phosphateShenggong Biological Engineering Co., Ltd, China
Propan-2-olHuihai Keyi Technology Co., Ltd, China
SDSSigma, China
Sodium chlorideShenggong Biological Engineering Co., Ltd, China
Sodium cyanoborohydride (NaBH3CN)Sigma, China
SpectrophotometerShanghai Yuanxi instrument Co., LtdB-500
Transfer liquid gunSmer Fell Science and Technology Co., Ltd, China4672100
TrisAmresco, America
Ultra-low temperature refrigeratorThermo Co., Ltd, AmericaMLT-1386-3-V; MDF-382E
Ultra-performance liquid chromatographyWatts technology Co., Ltd, ChinaAcquity MLtraPerformance LC
Vacuum PumpWatts technology Co., Ltd, China725000604
Volatilizing machine/DryerEppendorf Co., Ltd, GermanyT_1087461900
VortexChangzhou Enpei instrument Co., Ltd, ChinaNP-30S
Water-bathTester instruments Co., LtdDK-98-IIA
Weighing balanceShanghai Jingke Scientific Instrument Co., Ltd.MP200B

References

  1. Kolarich, D., Lepenies, B., Seeberger, P. H. Glycomics, glycoproteomics and the immune system. Current Opinion in Chemical Biology. 16, 214 (2012).
  2. Cummings, R., Pierce, J. M. The Challenge and Promise of Glycomics. Chemistry Biol....

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