免疫球蛋白 G N-甘油分析通过超高性能液色谱分析
Summary
免疫球蛋白G（IgG）N-甘油采用亲水相互作用色谱UPLC的特征是。 此外，IgG N-甘油的结构明显分离。这里介绍这个实验方法，以便它可以在研究环境中广泛使用。
Abstract
Glycomics 是组学系统研究的一个新的子专业，在发现下一代疾病易感性、药物靶点发现和精密医学的生物标志物方面具有巨大潜力。替代IgG N-甘油已报告在几个常见的慢性疾病，并建议在临床应用（即，诊断和预测疾病的生物标志物）有很大的潜力。IgG N-甘油采用亲水相互作用色谱法（HILIC）超高性能液相色谱（UPLC）进行广泛描述。UPLC是一种稳定的检测技术，具有良好的可重复性和较高的相对定量精度。此外，IgG N-甘甘的结构明显分离，血浆中的甘油成分和相对丰度具有特征。
Introduction
人类蛋白质的N-糖基化是一种常见和必要的转化后修饰1，有助于相对准确地预测疾病的发生和发展。由于其结构的复杂性，预计将有超过5000个甘油结构，为疾病2提供诊断和预测生物标志物的巨大潜力。附于免疫球蛋白G（IgG）的N-甘油已被证明对IgG的功能至关重要，IgG N-糖基化参与亲炎和抗炎系统之间的平衡3。差异IgG N-糖基化参与疾病发展和进展，代表疾病病理学中涉及的易感性和功能机制。IgG N-糖基化的炎症作用与衰老、炎症性疾病、自身免疫性疾病和癌症4相关。
随着检测技术的发展， 以下方法在高通量胶质中应用最广泛：亲水相互作用色谱（HILIC）具有荧光检测（UPLC-FLR）的高性能液相色谱，用激光诱导荧光的多路毛细管凝胶电泳 电感检测（xCGE-LIF）、矩阵辅助激光解吸/电光飞行质谱仪（MALDI-TOF-MS）和液相色谱电喷雾质谱（LC-ESI-MS）.这些方法克服了以前低通量、结果不稳定、灵敏度特异性差等缺点。
UPLC被广泛用于探索IgG N-糖基化与某些疾病（即衰老7、肥胖8、血脂异常9、II型糖尿病10、高血压11、缺血性中风12和帕金森病13）之间的联系。与上述其他三种方法相比，UPLC 具有以下优势5、14。首先，它提供了一种相对定量分析方法，涉及总面积规范化的数据分析提高了每个样本的可比性。其次，设备成本和所需的专业知识相对较低，这使得实施和将糖基化生物标志物转化为临床应用变得更加容易。这里介绍UPLC，以便它可以更广泛地使用。
Protocol
协议中的所有科目均已获首都医科大学道德委员会批准。在研究开始时，每个科目都获得了书面知情同意。 1. IgG隔离 制备化学品包括结合缓冲液（磷酸盐缓冲盐水，PBS）：1x PBS（pH = 7.4），中和缓冲液：10x PBS（pH = 6.6-6.8），脱液：0.1M甲酸（pH = 2.5），中和溶液的增色剂：1M碳酸氢铵，储存缓冲液：20%乙醇 = 20 mM Tris = 0.1 M NaCl （pH = 7.4），蛋白质 G 的?…
Representative Results
如图1所示，IgG N-甘油根据峰值位置和保留时间分析为24初始IgG甘油峰（GP）。根据先前的研究（表1）15，N-甘油结构可通过质谱检测获得。 为了确保结果具有可比性，应用了总面积正常化，其中每个峰值中的甘油量以总综合面积的百分比表示。 为了评估该方法?…
Discussion
UPLC作为相对定量分析方法5，15。结果表明，UPLC是一种稳定的检测技术，具有良好的可重复性和相对定量精度。每个峰值中的甘油量以 UPLC（相对值）的总集成面积的百分比表示。相对定量提高了测试样品的可比性。此外，96孔蛋白G板用于一次用96个样品来纯化IgG，用于高通量检测。蛋白质G结合IgG的能力大于蛋白质A的能力，如先前研究15、1…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（81673247和81872682）和澳大利亚-中国合作赠款（NH&MRC – APP1112767-NSFC 8156128020）的资助。
Materials
| 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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Cite This Article
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).