4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于<i>N</i>-糖肽特异性富集

引用本文: 张汉卿, 秦伟捷, 张养军. 4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集[J]. 色谱, 2020, 38(8): 891-899. doi: 10.3724/SP.J.1123.2020.01014 shu

Citation: ZHANG Hanqing, QIN Weijie, ZHANG Yangjun. Preparation of 4-mercaptophenylboronic acid functionalized two-dimensional molybdenum disulfide nanocomposite and its specific enrichment for N-glycopeptide[J]. Chinese Journal of Chromatography, 2020, 38(8): 891-899. doi: 10.3724/SP.J.1123.2020.01014 shu

4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集

1.
安徽医科大学研究生院, 安徽合肥 230032;
2.
军事医学研究院生命组学研究所, 北京蛋白质组研究中心, 蛋白质组学国家重点实验室, 北京 102206

通讯作者: 张养军, E-mail:13683167093@163.com

基金项目:
国家重点研发计划（2017YFA0505002，2018YFC0910302，2016YFA0501403，2018YFF0212505）；国家自然科学基金（21675172）；蛋白质组学国家重点实验室（SKLP-K201706）；北京市泌尿细胞分子诊断重点实验室（2019-KF21）.

摘要: 蛋白质的N-糖基化修饰在多种生物过程中发挥着至关重要的作用，近年来的许多研究证实异常的蛋白质糖基化与多种疾病的发生发展密切相关，表明糖基化蛋白质具有较大的潜力成为新的生物标志物或者药物靶标。在样本的处理过程中，对N-糖基化肽段进行富集分离后再进行质谱分析已经成为糖蛋白质组学分析前的必要步骤。但是，由于复杂生物样本中N-糖基化肽段的丰度低和离子化效率差等问题，通过质谱鉴定N-糖肽仍然是一项艰巨的任务。研究通过将纳米金线（Au）、4-巯基苯硼酸（4-MPB）与超薄二维二硫化钼（2D-MoS₂）进行反应，成功制备了一种用于富集蛋白质N-糖基化肽段的新型功能纳米复合材料（MoS₂/Au/4-MPB）。二硫化钼纳米材料的层状结构可以为反应提供大量的可修饰位点，便于修饰纳米金线；功能基团4-巯基苯基硼酸对N-糖肽具有高度的选择性，可以对生物样品中N-糖基化肽段进行特异性富集。使用标准蛋白人免疫球蛋白G（IgG）和牛血清白蛋白（BSA）胰蛋白酶酶切产物对新型功能纳米材料的N-糖基化肽段的富集性能进行评估，其灵敏度达到5 fmol，选择性达到1：1000。将其用于生物样品中N-糖基化肽段的富集，从50 μg尿液外泌体蛋白胰蛋白酶酶切产物中共富集鉴定出768个N-糖肽，归属于377个蛋白质。这些结果表明该新型功能纳米复合材料对复杂生物样品中N-糖肽的选择富集有着巨大的应用潜力，为糖蛋白质组的研究提供了一种新方法。

关键词:

English

Preparation of 4-mercaptophenylboronic acid functionalized two-dimensional molybdenum disulfide nanocomposite and its specific enrichment for N-glycopeptide

1.
Graduate School, Anhui Medical University, Hefei 230032, China;
2.
Beijing Institute of Lifeomics, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing 102206, China

Corresponding author: ZHANG Yangjun, 13683167093@163.com

Received Date: 14 January 2020
Fund Project:
National Key Program for Basic Research of China (Nos. 2017YFA0505002, 2018YFC0910302, 2016YFA0501403, 2018YFF0212505)

Abstract: Protein N-glycosylation plays a crucial role in the folding, transportation, and localization of proteins, and participates in many important biological processes such as receptor activation and signal transduction. An increasing number of studies have shown that abnormal protein glycosylation is closely related to various diseases. Therefore, N-glycosylated proteins are potential candidates for become new biomarkers or drug targets. The current research strategy for N-glycosylated proteins is to first digest the protease into peptides and then identify them by mass spectrometry. In sample preparation processing, enrichment and separation of N-glycopeptides have become the vital step for glycoproteomic analysis. However, because of their low abundance and poor ionization, mass spectrometric identification of N-glycopeptides in complex samples remains a challenging task. Therefore, the development of a selective enrichment strategy for N-glycopeptides from complex biological samples is necessary. In this work, new functional nanomaterials were prepared through a simple, convenient, and efficient two-step "Au-S" reaction. In short, the "Au-S" bond was first used to load nano-gold wires on the surface of molybdenum disulfide (MoS₂), and then to connect these wires to 4-mercaptophenylboronic acid (4-MPB). The resulting nanomaterial named MoS₂/Au/4-MPB was successfully prepared by serial functionalization of ultra-thin two-dimensional MoS₂, nano-gold wires and 4-MPB for the efficient enrichment of N-glycopeptides. The layered structure of molybdenum disulfide nanomaterials can provide numerous modifiable sites for the reaction, allowing for convenient modification of the nano-gold wires. The functional group 4-MPB has high affinity for N-glycopeptides, and it can selectively enrich them in biological samples. In order to evaluate the N-glycopeptide enrichment performance of MoS₂/Au/4-MPB, standard proteins human immunoglobulin G (IgG) and bovine serum albumin (BSA) trypsin digests were used. Thus, facilitated interactions with N-glycopeptides for boric acid-based retention could be expected. Low femtomolar detection sensitivity, 1:1000 enrichment selectivity, and 100 μg/mg loading capacity were achieved for N-glycopeptide enrichment. In the application of this material to biological samples, exosomes were chosen as the research objects. On the one hand, exosomes serve as a new class of carriers for intercellular communication and drug delivery. Almost all types of cells can secrete exosomes. On the other hand, urine is another potential source for liquid biopsy samples in addition to blood. Obtaining urine samples is a less invasive process, and urine samples are easier to handle and save as compared to blood samples. Exosomes can also be detected in urine. Cell-specific components such as proteins, lipids, and nucleic acids can provide diverse information, which is of great significance in disease diagnosis and treatment. However, there are only a few studies on urinary exosomes N-glycoprotein group. In this research, N-glycoproteins were successfully identified in urine exosomes. In three replicate experiments, 536, 515 and 487 N-glycosylated peptides were identified from 279, 270 and 279 N-glycoproteins, respectively. After de-duplication of the three sets of data, a total of 768 N-glycopeptides corresponding to 377 N-glycoproteins were identified. This result indicates that the novel nanocomposite has good enrichment selectivity and sensitivity for N-glycopeptides enrichment in complex biological samples, and this method provides a new method for glycoproteomics research.

Key words:

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4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集

通讯作者: 张养军, E-mail:13683167093@163.com

English

Preparation of 4-mercaptophenylboronic acid functionalized two-dimensional molybdenum disulfide nanocomposite and its specific enrichment for N-glycopeptide

Corresponding author: ZHANG Yangjun, 13683167093@163.com

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集

通讯作者: 张养军, E-mail:13683167093@163.com

English

Preparation of 4-mercaptophenylboronic acid functionalized two-dimensional molybdenum disulfide nanocomposite and its specific enrichment for N-glycopeptide

Corresponding author: ZHANG Yangjun, 13683167093@163.com

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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通讯作者: 陈斌, bchen63@163.com

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