Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

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.....

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 method^5,15. 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.......

Materials

Name	Company	Catalog Number	Comments
2-aminobenzamide, 2-AB	Sigma, China
96-well collection plate	AXYGEN
96-well filter plate	Pol	0.45 um GHP
96-well monolithic plate	BIA Separations
96-well plate rotor	Eppendorf Co., Ltd, Germany	T_1087461900
Acetic acid	Sigma, China
Acetonitrile	Huihai Keyi Technology Co., Ltd, China
Ammonium bicarbonate	Shenggong Biological Engineering Co., Ltd, China
Ammonium formate	Beijing Minruida Technology Co., Ltd.
Constant shaking incubator/rocker	Zhicheng analytical instrument manufacturing co., Ltd, China	ZWY-10313
Dextran Calibration Ladder/Glycopeptide column	Watts technology Co., Ltd, China	BEH column
Dimethyl sulfoxide (DMSO)	Sigma, China
Disodium phosphate	Shenggong Biological Engineering Co., Ltd, China
Electric ovens	Tester instruments Co., Ltd	202-2AB
Empower 3.0	Waters technology Co., Ltd, America
Ethanol	Huihai Keyi Technology Co., Ltd, China
Formic acid	Sigma, China
GlycoProfile 2-AB Labeling kit	Sigma, China
HCl	Junrui Biotechnology Co., Ltd, China
High-speed centrifuge	Eppendorf Co., Ltd, Germany	5430
Igepal	Sigma, China
Low temperature centrifuge	Eppendorf Co., Ltd, Germany
Low temperature refrigerator	Qingdao Haier Co., Ltd
Manifold 96-well plate	Watts technology Co., Ltd, China	186001831
Methanol	Huihai Keyi Technology Co., Ltd, China
Milli-Q pure water meter	Millipore Co., Ltd, America	Advantage A10
NaOH	Shenggong Biological Engineering Co., Ltd, China
PH tester	Sartorius Co., Ltd, Germany	PB-10
Phosphate buffered saline, PBS	Shenggong Biological Engineering Co., Ltd, China
Pipette	Eppendorf Co., Ltd, Germany	4672100, 0.5-10μl & 10-100μl & 20-200μl & 1000μl
PNGase F enzyme	Sigma, China
Potassium dihydrogen phosphate	Shenggong Biological Engineering Co., Ltd, China
Propan-2-ol	Huihai Keyi Technology Co., Ltd, China
SDS	Sigma, China
Sodium chloride	Shenggong Biological Engineering Co., Ltd, China
Sodium cyanoborohydride (NaBH3CN)	Sigma, China
Spectrophotometer	Shanghai Yuanxi instrument Co., Ltd	B-500
Transfer liquid gun	Smer Fell Science and Technology Co., Ltd, China	4672100
Tris	Amresco, America
Ultra-low temperature refrigerator	Thermo Co., Ltd, America	MLT-1386-3-V; MDF-382E
Ultra-performance liquid chromatography	Watts technology Co., Ltd, China	Acquity MLtraPerformance LC
Vacuum Pump	Watts technology Co., Ltd, China	725000604
Volatilizing machine/Dryer	Eppendorf Co., Ltd, Germany	T_1087461900
Vortex	Changzhou Enpei instrument Co., Ltd, China	NP-30S
Water-bath	Tester instruments Co., Ltd	DK-98-IIA
Weighing balance	Shanghai Jingke Scientific Instrument Co., Ltd.	MP200B