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

* These authors contributed equally
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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.

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Liu, D., Xu, X., Li, Y., Zhang, J., Zhang, X., Li, Q., Hou, H., Li, D., Wang, W., Wang, Y. Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography. J. Vis. Exp. (155), e60104, doi:10.3791/60104 (2020).

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 specificity5,6.

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 advantages5,14. 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.

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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:

      CIgG = 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.

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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 of the total integrated area.

To assess the repeatability and stability of the method, the standard sample was tested in parallel six times. As shown in Table 2, the coefficient of variation (CV) of 24 GPs ranged from 1.84%-16.73%, 15 (62.50%) of which were below 10%. GPs with relatively small proportions (≤1.16%) showed high measurement errors (more than 10% of CV). In addition, the IgG N-glycan profiles combined from 76 individuals (Figure 2) indicated that the position of GP was stable, shape of GP was similar, and integration for the samples maintained the same intervals. The above results indicate that the method is stable and repeatable.

As shown in Table 3, an additional 36 derived traits describing the relative abundances of galactosylation, sialylation, bisecting GlcNAc, and core fucosylation were calculated by the remaining 24 directly measured glycans. For example, G2/G0 (GP12/GP2) reflected the level of galactosylation (di-/a-) without core fucosylation and bisecting GlcNAc. G2/G1 (GP14/[GP8 + GP9]) reflected the level of galactosylation (di-/mono-) with core fucosylation and without bisecting GlcNAc. Finally, G1/G0 ([GP10 + GP11]/GP6) reflected the level of galactosylation (mono-/a-) with core fucosylation and bisecting GlcNAc. These calculations of derived glycans follow a principle, to see the change of only one glycosylation trait.

Figure 1
Figure 1: UPLC chromatogram of one individual. IgG N-glycans were analyzed into 24 initial IgG glycan peaks (GPs) based on peak position and retention time. GP8 represents the sum of GP 8a and GP 8b. GP16 represents the sum of GP 16a and GP 16b. The structure of glycans in each chromatographic peak and the average percentage of individual structures are shown in Table 1 and Table 2. Please click here to view a larger version of this figure.

Figure 2
Figure 2: UPLC chromatogram of 76 individuals. The IgG N-glycan profiles were combined from 76 individuals to demonstrate the repeatability and stability of the method. Please click here to view a larger version of this figure.

Table 1
Table 1: Structure of the 24 initial IgG glycans. GP: glycan peak; F: fucose; A: number of antenna's attached to the core sequence (existing of two NAcetylglucosamine (GlcNAc) and three mannose residues); B: bisecting GlcNac; G: galactose; S: sialic acid. Structural schemes are defined as follows: blue square: GlcNac; green circle: mannose; red triangle: core fucose/ antennary fucose; yellow circle: galactose; purple rhomb: sialic acid.

Glycan peak Mean (SD) CV (%) Glycan peak Mean (SD) CV (%)
GP1 0.23 (0.017) 7.28 GP13 0.50 (0.043) 8.64
GP2 0.24 (0.034) 13.97 GP14 19.54 (0.36) 1.84
GP3 0.96 (0.16) 16.73 GP15 1.16 (0.14) 11.63
GP4 19.52 (0.58) 2.98 GP16 2.79 (0.19) 6.93
GP5 0.17 (0.024) 13.86 GP17 1.29 (0.13) 9.78
GP6 3.19 (0.26) 8.22 GP18 13.16 (0.51) 3.85
GP7 0.29 (0.033) 11.51 GP19 2.12 (0.21) 9.76
GP8 16.45 (0.62) 3.77 GP20 0.12 (0.018) 14.91
GP9 8.97 (0.52) 5.74 GP21 1.05 (0.17) 16.09
GP10 2.97 (0.22) 7.34 GP22 0.18 (0.025) 14.16
GP11 0.30 (0.041) 13.82 GP23 2.14 (0.14) 6.45
GP12 1.27 (0.026) 2.08 GP24 1.43 (0.13) 9.12

Table 2: Precision of the method. The standard sample is tested in parallel six times to assess the repeatability and stability of the method. CV: Coefficient of Variation; GP: Glycan peak; SD: Standard deviation.

Derived glycans Formulas Derived glycans Formulas
Galactosylation Fucosylation
G2/G0 GP12/GP2 F1/F0 GP4/GP2
GP14/GP4 GP6/GP3
GP15/GP6 (GP8+ GP9)/GP7
G2/G1 GP12/GP7 GP14/FGP12
GP14/(GP8+ GP9) GP15/GP13
GP15/(GP10+ GP11) GP18/GP17
G1/G0 GP7/GP2 GP23/GP21
(GP8+ GP9)/GP4 GP24/GP22
(GP10+ GP11)/GP6
Sialylation Bisecting GlcNAc
S2/S0 GP21/GP12 B1/B0 GP3/GP2
GP22/GP13 GP6/GP4
GP23/GP14 (GP10+GP11)/ (GP8+ GP9)
GP24/GP15 GP13/GP12
S2/S1 GP21/GP17 GP15/GP14
GP23/GP18 GP19/GP18
GP24/GP19 GP22/GP21
S1/S0 GP17/GP12 GP24/GP23
GP18/GP13
GP16/GP14
GP19/GP15

Table 3: Calculation of the derived glycans. GP: glycan peak; F: fucose; B: bisecting GlcNac; G: galactose; S: sialic acid.

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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 samples at one time for high throughput detection. The ability of protein G to bind IgG is greater than that of protein A, as described in previous studies15,16.

In addition, the structures of IgG N-glycan are clearly separated. The derived IgG glycans describe the level of galactosylation, sialylation, bisecting GlcNAc, and core fucosylation, which are calculated by the initial IgG N-glycans. In a previous study, some derived glycans (FBn, FBG0n / G0n, FBn / Fntotal, Bn / (Fn + FBn), FBG2n / FG2n, FG2n / (BG2n + FBG2n), BG2n / (FG2n + FBG2n)) could reflect the change in multiple glycosylation levels but did not reflect the level of specific glycosylation15.

Recently, a large-scale study showed that IgG galactosylation (referred to as Gal-ratio) can serve as a promising biomarker for cancer screening in multiple cancer types17. The distribution of IgG galactosylation is measured by calculating the relative intensities of agalactosylated (G0) vs. monogalactosylated (G1) and digalactosylated (G2) N-glycans according to the formula (G0/[G1 + G2 x 2]). The Gal-ratio reflects the level of galactosylation with core fucosylation and without bisecting GlcNAc. Therefore, multiple initial IgG N-glycans were combined with a derived glycan, representing the level of a specific glycosylation trait. The calculations of derived glycans follow a principle that explores the changes in only one glycosylation trait fixed over other glycosylation trait.

In the present protocol, pH is important for maintaining the stability of IgG and glycan structures, especially for stabilizing terminal sialylation. Therefore, the pH value of the solution must be strictly controlled, and the pH of the solution exposed to IgG must be restored as well as glycans kept at a neutral pH level. In addition, the glycan release step is critical in this protocol. The key to glycan release is improving the activity of the PNGase F enzyme to maximize its efficiency. For example, it was found that 18-20 h is optimal for PNGaseF digestion. This step needs to be fully reacted.

There are some limitations of this technique. The method used cannot differentiate glycans released from the Fab and the Fc portions of IgG. Glycans from Fab and Fc are known to be different. With the developments in glycoproteomics, detection techniques can measure the levels of IgG combining N-glycans to explore the role of IgG N-glycans and N-glycosylation in diseases. The cost of equipment is relatively low; however, the cost per sample is rather high.

In summary, this protocol introduces UPLC so that it can be widely used. Comprehensive valuation and standardization of the analytical methods are needed before significant amounts of time and resources are invested in large-scale studies. As UPLC becomes more widely used, the effects of IgG N-glycans and N-glycosylation on certain diseases can be more accurately determined, and glycosylation biomarkers can be used for clinical applications.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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

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

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References

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