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. Differential IgG N-glycosylation is involved in disease development and progression, representing both a predisposition and functional mechanism involved in disease pathology. The inflammatory role of IgG N-glycosylation has been associated with aging, inflammatory diseases, autoimmune diseases, and cancer⁴.

With the development of detection technology, the following methods are most widely used in high throughput glycomics: hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR), multiplex capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography electrospray mass spectrometry (LC-ESI-MS). These methods have overcome previous shortcomings of low flux, unstable results, and poor sensitivity specificity^5,6.

UPLC is widely used to explore the association between IgG N-glycosylation and certain diseases (i.e., ageing⁷, obesity⁸, dyslipidemia⁹, type II diabetes¹⁰, hypertension¹¹, ischemic stroke¹², and Parkinson's disease¹³). Compared to the other three abovementioned methods, UPLC has the following advantages^5,14. First, it provides a relative quantitative analysis method, and the data analysis that involves total area normalization improves the comparability of each sample. Second, the cost of equipment and required expertise are relatively low, which makes it easier to implement and transform glycosylation biomarkers into clinical applications. Presented here is an introduction to UPLC so it can be more widely used.

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.
   NOTE: The level of pH is critical in this protocol. The elution of IgG requires a very low pH, and there is a risk of the loss of sialic acids due to acid hydrolysis. Therefore, elution occurs within seconds, and the pH is quickly restored to neutrality, preserving the integrity of IgG and the sialic acids.
2. Prepare the samples: thaw the frozen plasma sample then centrifuge at 80 x g for 10 min, and leave the protein G monolithic plate and the abovementioned chemicals for 30 min at room temperature (RT).
3. Transfer a 100 µL sample (which can be used to detect 2x to prevent the first failure) into a 2 mL collection plate (here, a total of six standard samples, one control sample [ultra-pure water], and 89 plasma samples were designed for 96 well plates and randomly assigned to the plate).
4. Dilute the samples with 1x PBS by 1:7 (v/v).
5. Clean a 0.45 µm hydrophilic polypropylene (GHP) filter plate with 200 µL of ultra-pure water (repeat 2x).
6. Transfer the diluted samples into the filter plate and filter the samples into the collection plate using a vacuum pump (control vacuum pressure at 266.6-399.9 Pa).
7. Preparation of the protein G monolithic plates
   1. Discard the storage buffer.
   2. Clean the monolithic plates with 2 mL of ultra-pure water, 2 mL of 1x PBS, 1 mL of 0.1 M formic acid, 2 mL of 10x PBS, 2 mL of 1x PBS (sequentially), and remove flowing liquid using a vacuum pump.
8. Transfer the filtered samples to the protein G monolithic plate for IgG binding and cleaning, then clean the monolithic plates with 2 mL of 1x PBS (repeat the cleaning 2x).
9. Elute IgG with 1 mL of 0.1 M formic acid and filter the samples into the collection plate by vacuum pump, then add 170 µL of 1 M ammonium bicarbonate into the collection plate.
10. Detect IgG concentration using an absorption spectrophotometer (optimal wavelength = 280 nm).
    1. Open the software and select the protein-CY3 mode.
    2. Draw 2 µL of ultra-pure water and load it into the screen, then click Blank in the software to clear the screen (repeat 1x).
    3. Draw 2 µL of ultra-pure water and load it into the screen, then click Sample in the software to detect the ultra-pure water.
    4. Draw 2 µL of IgG sample and load it into the screen, then click Sample in the software to detect the sample.
    5. Draw 2 µL of ultra-pure water and load it into the screen, then click Blank in the software to clear the screen.
    6. Close the software.
       NOTE: The formula for calculating IgG concentration is as follows:
       
       C_IgG = absorbance x extinction coefficient (13.7) x 1,000 µg/mL
11. Put the extracted IgG to dry in an oven at 60 °C and preserve the extracted IgG (300 µL extracted IgG for 4 h).
    1. Remove 300 µL of extracted IgG if the concentration is greater than 1,000 µg/mL.
    2. Remove 350 µL of extracted IgG if the concentration is between 500-1,000 µg/mL.
    3. Remove 400 µL of extracted IgG if the concentration is between 200-500 µg/mL.
    4. Remove 600 µL of extracted IgG if the concentration is smaller than 200 µg/mL.
       NOTE: The concentration of IgG should be preferably >200 µg/mL for subsequent detection. The average amount of IgG should be preferably >1,200 µg, which can be tested 2x in case the first test fails.
12. Cleaning the protein G monolithic plate for reuse
    1. Wash the plate with 2 mL of ultra-pure water, 1 mL of 0.1 M NaOH (for removing precipitated proteins), 4 mL of ultra-pure water, and 4 mL of 1x PBS (sequentially), then remove flowing liquid using a vacuum pump.
    2. Wash the plate with 2 mL of ultra-pure water, 2 mL of 30% propan-2-ol (for removing bound hydrophobic proteins), 2 mL of ultra-pure water, and 4 mL of 1x PBS (sequentially), then remove flowing liquid using a vacuum pump.
    3. Wash the plate with 1 mL of buffer (20% ethanol + 20 Mm Tris + 0.1 M NaCl) and add 1 mL of buffer (20% ethanol + 20 Mm Tris + 0.1 M NaCl) to the plate, then leave the plate at 4 °C.

2. Glycan release

1. Prepare the dried IgG and store the chemicals including 1.33% SDS, 4% Igepal (store away from light), and 5x PBS at RT.
2. Prepare PNGase F enzyme by diluting 250 U enzyme with 250 µL of ultra-pure water.
3. Denaturation of IgG
   1. Add 30 µL of 1.33% SDS and mix by vortexing, transfer the sample into a 65 °C oven for 10 min, then remove it from the oven and let rest for 15 min.
   2. Add 10 µL of 4% Igepal and place it on the shaking incubator for 5 min.
4. Removal and release of glycans
   1. Add 20 µL of 5x PBS and 30-35 µL of 0.1 mol/L NaOH to regulate a pH of 8.0, and mix by vortexing. Add 4 µL of PNGase F enzyme and mix by vortexing. Then, incubate for 18-20 h in a 37 °C water bath.
   2. Put the released glycans to dry in an oven at 60 °C for 2.5-3.0 h.
   3. Save the released glycans at -80 °C until further measurement.
      NOTE: This step is critical. The key to glycan release is improving the activity of the PNGase F enzyme to maximize its efficiency.

3. Glycan labeling and purification

1. Prepare the 2-aminobenzamide (2-AB) labeling reagent with 0.70 mg of 2-AB, 10.50 µL of acetic acid, 6 mg of sodium cyanoborohydride (NaBH₃CN), and 24.50 µL of dimethyl sulfoxide (DMSO) (total volume = 35 µL). Then, add acetic acid, 2-AB, and NaBH₃CN into the DMSO in order.
2. Label the glycans using 35 µL of 2-AB labeling reagent, transfer the labeled glycans to the oscillator for 5 min, transfer to the oven for 3 h at 65 °C, then transfer to RT for 30 min.
   NOTE: The entire glycan labeling step must be performed while protected from light.
3. Pretreat a 0.2 µm GHP filter plate with 200 µL of 70% ethanol, 200 µL of ultra-pure water, and 200 µL of 96% acetonitrile (4 °C), then remove waste using a vacuum pump.
4. Purification of 2-AB labeled glycan
   1. Add 700 µL of 100% acetonitrile to the 2-AB labeled glycan and transfer to a shaking incubator for 5 min.
   2. Centrifuge at 134 x g for 5 min (4 °C).
   3. Transfer the sample to a 0.2 µm GHP filter plate for 2 min and remove the filtrate (flowing liquid) using a vacuum pump.
5. Wash 2-AB labeled glycan with 200 µL of 96% acetonitrile (4 °C) and remove the filtrate (flowing liquid) using a vacuum pump 5x-6x.
6. Elute 2-AB labeled glycan with 100 µL of ultra-pure water 3x.
7. Transfer the 2-AB labeled glycan into an oven to dry at 60 °C for 3.5 h.
8. Save the labeled N-glycans at -80 °C until further measurement.

4. Hydrophilic interaction chromatography and analysis of glycans

1. Conditioning of UPLC instruments and preparation of mobile phases
   1. Prepare mobile phases including solvent A: 100 mM ammonium formate (pH = 4.4), solvent B: 100% acetonitrile, solvent C: 90% ultra-pure water (10% methanol), and solvent D: 50% methanol (ultra-pure water).
   2. Open the software to control the mobile phases.
   3. Wash UPLC instruments at flow rate of 0.2 mL/min (50% solvent B and 50% solvent C) balancing for 30 min, then at a flow rate of 0.2 mL/min (25% solvent A and 75% solvent B) balancing for 20 min, then a flow rate of 0.4 mL/min balancing.
2. Dissolve the labeled N-glycans with 25 µL of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 ratio (v/v). Then, centrifuge at 134 × g for 5 min (4 °C) and load 10 µL of the labeled N-glycans into the UPLC instruments.
3. Separate the labeled N-glycans at flow rate of 0.4 mL/min with a linear gradient of 75% to 62% acetonitrile for 25 min. Then, perform an analytical run by dextran calibration ladder/glycopeptide column on a UPLC at 60 °C (here, samples were kept at 4 °C prior to injection).
4. Detect N-glycan fluorescence at excitation and emission wave lengths of 330 nm and 420 nm, respectively.
5. Integrate the glycans based on peak position and retention time.
6. Calculate the relative value of each Glycan Peak (GP)/ all Glycan Peaks (GPs) (percentage, %) as follows: GP1: GP1/GPs*100, GP2: GP2/GPs*100, GP3: GP3/GPs*100, etc.

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

List of materials used in this article

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