色谱

色谱 ›› 2019, Vol. 37 ›› Issue (3): 279-286.DOI: 10.3724/SP.J.1123.2018.11015

• 研究论文 • 上一篇    下一篇

基于叠氮基-氰基点击化学的苯硼酸亲和硅胶的制备及其在糖蛋白/糖肽选择性富集中的应用

张丽媛1, 王立恒2, 王丽莉1, 董佩佩1, 刘和真1, 赵艳艳1   

  1. 1. 大连医科大学, 辽宁 大连 116044;
    2. 大连市第二人民医院骨科康复中心, 辽宁 大连 116001
  • 收稿日期:2018-11-09 出版日期:2019-03-08 发布日期:2015-01-21
  • 通讯作者: 赵艳艳.Tel:86110113,E-mail:zhaoyanyan917@163.com.
  • 基金资助:

    国家自然科学基金（21105007，21505015）；辽宁省自然科学基金（20170540286）；大连市支持高层次人才创新创业计划（2016RQ041）；辽宁省教育厅科学研究重点项目（LZ2016001）.

Preparation of novel phenyl boronic acid functionalized silica gel using triazo-cyanide click chemistry and its application in glycoprotein/glycopeptide selective enrichment

ZHANG Liyuan1, WANG Liheng2, WANG Lili1, DONG Peipei1, LIU Hezhen1, ZHAO Yanyan1   

  1. 1. Dalian Medical University, Dalian 116044, China;
    2. Orthopaedic Rehabilitation Center, the Second Hospital of Dalian, Dalian 116001, China
  • Received:2018-11-09 Online:2019-03-08 Published:2015-01-21
  • Supported by:

    National Natural Science Foundation of China (Nos. 21105007, 21505015); Liaoning Provincial Natural Science Foundation of China (No. 20170540286); Dalian High-Level Talents Innovation Support Program (No. 2016RQ041); Program of the Key Lab in Liaoning Province Education Department (No. LZ2016001).

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摘要：

硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性，生物相容性，制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题，设计并开发了一种新型苯硼酸亲和硅胶（TCNBA）。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成，生物相容性好，制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行评价，结果表明，TCNBA能够分别从辣根过氧化物酶（HRP）和免疫球蛋白G（IgG）酶解液中鉴定出13个和11个糖肽；以HRP和牛血清白蛋白（BSA）酶解液混合物（物质的量比1：10）作为研究对象，富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）进行评价，以HRP、IgG、核糖核酸酶B（RNaseB）作为考察对象，结果表明，TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示，富集后非糖蛋白得到较大程度去除，糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。

关键词: 分离, 硼亲和色谱, 糖蛋白, 糖肽, 选择性富集

Abstract:

The application of boronic acid affinity chromatography to glycoprotein/glycopeptide enrichment is increasingly maturing. The enrichment selectivity, biocompatibility, and facile operation protocol are key aspects in efficient enrichment methods. In this work, a novel triazo-cyanide boronic acid functionalized material (TCNBA) was prepared using triazo-cyanide click chemistry. The TCNBA was proved to be successfully synthesized through infrared ray (IR) characterization. Subsequently, the glycopeptide/glycoprotein enrichment selectivity of the TCNBA was evaluated. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF MS) was employed for the glycopeptide enrichment selectivity evaluation. Taking the digestion of horseradish peroxidase (HRP) and immunoglobulin G (IgG) as samples, 13 and 11 glycopeptides could be characterized with improved signals after TCNBA enrichment, respectively. High abundance non-glycopeptides could be removed effectively from the eluting fraction. This result indicates the high glycopeptide enrichment selectivity of TCNBA. In addition, a mixture of HRP and bovine serum albumin (BSA) enzymatic solution (1:10, amount of substance ratio) was utilized as a sample, and five glycopeptide signals could be identified following enrichment. To evaluate the glycoprotein enrichment selectivity, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) was adopted as an evaluation method. Mixtures of HRP, IgG, BSA, and ribonuclease B (RNaseB) proteins were employed as samples, and the results demonstrated that TCNBA had a high glycoprotein enrichment selectivity. The application of TCNBA to the analysis of a real biosample was also evaluated using human plasma. The results indicated the TCNBA could be utilized in large-scale glycoprotein analysis.

Key words: boronic acid affinity chromatography, glycopeptides, glycoprotein, selective enrichment, separation

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