水溶性硼酸亲和材料的制备及在肺癌转移预警的糖蛋白质分析中的应用

Almost all of the current isolation methods are based on solid phase extractions, which, due to the nature of the heterogeneous environment and nonlinear binding dynamics when dealing with extremely low abundance glycoproteins, can yield poor reproducibility, low recovery and inferior sensitivity. Moreover, it is difficult to obtain high-quality glycol-specific antibodies. To address these issues, in this project, a novel multi-functionalized soluble material for the enrichment and detection of glycoprotein is devised based on polyamidoamine dendrimers (PAMAM) and boronic acid affinity technology, and will be applied in lung cancer EMT related glycoproteome research. The PAMAM with excellent solubility, controlled surface functionalities is modified with boronic acid to capture glycoproteins in a homogeneous environment, which could obtain an outstanding reproducibility and high recovery, compared with biased and unreproducible solid phase based strategies. Subsequently a biotin group is modified on PAMAM, which allow for isolation or detection of glycoproteins with the high specific Biotin-Avidin-System. This dendrimeric soluble material could be used to trap glycoproteins in biological sample as the sample pretreatment material prior to MS analysis, or to replace the traditional glycol-specific antibodies in immunoblotting, ELISA, SDS-PAGE for qualitative and quantitative assessment of protein glycosylation. This project could not only provide new insight into developing new isolation methods for glycoproteins, but also play an important role in revealing the mystery of EMT at the protein level, early diagnosis and treatment of lung cancer.

针对基于固相材料的糖蛋白富集分离方法存在的重现性差、回收率低、灵敏度差以及高质量的糖蛋白抗体难以制备等问题，本项目拟发展基于树枝状聚合物PAMAM的多功能水溶性硼酸亲和新材料，并将其应用于肺癌EMT转移机制的糖蛋白质组分析。本研究结合PAMAM良好的水溶性、表面功能团易于修饰以及硼酸技术的特点，制备可在均相溶液中实现富集的新材料，有效避免了基于固相材料的富集不均一性，可显著提高富集分析的重现性和回收率；然后，通过修饰生物素基团，利用高特异性的亲和素实现目标糖蛋白的高效分离或高灵敏度定量分析。本项目研制的水溶性硼酸亲和材料可作为糖蛋白富集分离材料应用于质谱前的生物样品预处理，亦可代替传统生物抗体应用于免疫印迹、ELISA、SDS-PAGE等进行生物样品中糖蛋白的定性及定量分析。本研究为发展新的糖蛋白富集分离方法提供新思路，并且对在蛋白质水平揭示肿瘤转移机制，实现临床预警和诊治具有重要意义。

针对基于固相材料的糖蛋白富集分离方法存在的重现性差、回收率低、灵敏度差以及高质量的糖蛋白抗体难以制备等问题，本项目发展了基于树枝状聚合物PAMAM的多功能水溶性硼酸亲和新材料。该材料结合了PAMAM良好的水溶性、表面功能团易于修饰以及硼酸技术的特点，其对糖蛋白的检测灵敏度可达0.1ng,比文献报道方法的灵敏度提高了6000倍，对糖蛋白的选择性与文献相比提高了100倍左右。其在肺癌细胞模型、蛋清以及人肝微粒体等生物样品的糖蛋白质组分析结果证明该材料可代替传统生物抗体应用于免疫印迹、ELISA、SDS-PAGE等生物样品中糖蛋白的定性及定量分析。本研究结果为发展新的糖蛋白富集分离及检测方法提供新思路。

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