基于生物条码专属分离和放大-质谱检测标签增敏的体内蛋白多肽类药物定量分析新方法研究

The development and evaluation of protein and polypeptide drugs (PPD) are the hot spot in research of new drugs nowadays. It is necessary to determine the concentration of PPD in biological samples during the evaluation of new drugs. The methods currently used for determination of PPD in vivo have some disadvantages, such as enzyme-linked immunosorbent assay methods with narrow linear range, bad precision and low sensitivity. Besides, determination of PPD in biological samples by LC-MS directly also has the problems of difficult sample pretreatment process and low sensitivity. In view of the above problems, this project intends to combine bio-barcode and LC-MS to develop a new method for quantification of PPD. The antibodies on the surface of the magnetic beads probes and colloidal gold probes can recognize specifically the same PPD in the biological samples and form complexes. Then the complexes can be purified by magnetic field. Lastly, tags that are dissociated from the colloidal gold probes can be determined by LC-MS. Thus, the PPDs can be purified specifically from the biological samples, and the detectable amounts of targets PPD can be amplified by using more tags. The mass responses of the selected tags are much higher than that of the PPD, which enhance the sensitivity once again. So, this method could achieve high-sensitivity detection of PPD in biological samples. This study will provide a new method for the quantification of PPD in biological samples.

蛋白多肽类药物（PPD）的开发与评价是当今新药研究热点，测定生物样品中PPD浓度是其新药评价必不可少的环节。目前PPD体内分析所采用的酶联免疫法等技术存在线性范围窄、测定误差大等缺陷；采用LC-MS法直接分析体内PPD也存在样品处理困难，灵敏度低等问题。针对上述问题，本项目拟将“生物条码”和LC-MS两种技术相结合，研究一种PPD定量分析新方法。即采用磁珠探针和胶体金探针表面的抗体分别特异地识别生物样品中同一PPD，形成三者的复合物，再外加磁场使该复合物分离纯化，用LC-MS法测定该复合物中胶体金表面的标签分子。该方法可以从生物样品中专属地分离PPD，并以标签分子对PPD进行数量放大，以大幅度提高检测目标的数量；此外，再通过质谱响应远高于PPD的标签分子达到质谱增敏效果，实现二次提高PPD检测灵敏度的目的，从而实现生物样品中PPD的高灵敏度检测。本研究将为PPD体内定量测定提供新方法。

蛋白多肽类药物由于其高生物活性、低毒性等优点成为当今医药领域的热点，但针对蛋白多肽类药物的分析方法却未跟上脚步。目前蛋白多肽类药物采用的酶联免疫法等技术存在线性范围窄、测定误差大等缺陷，而采用灵敏度高、准确度好的LC-MS法直接分析蛋白多肽类药物也由于蛋白多肽类大分子分子量大，与质谱检测器离子源的兼容性较差，离子化效率偏低等原因，导致质谱响应极低。质谱标签是一类具有高质谱响应，可大量修饰在纳米材料表面，实现信号转化及信号放大的小分子。借助质谱标签、纳米材料等新分析技术，扩大质谱的应用范围，已经成为了质谱应用研究的热点。在本项目中，我们基于质谱标签、纳米材料、适配体等分析新技术，以凝血酶为示范性研究对象，开展高灵敏度、高特异性的蛋白多肽检测新方法研究。在研究过程中，我们以基金计划书为原型，参考蛋白多肽类大分子检测的最新研究进展，根据示范性研究对象凝血酶的性质特点，对研究方案进行了进一步的完善，开展了一系列的研究工作。本课题组从质谱标签信号转化增敏入手，结合纳米技术、适配体识别技术，以灵敏度、准确度、选择性、可靠性为考量，建立了几种效果显著且灵活多变、普适性好的信号转化及信号放大方法，实现了示范性研究对象凝血酶的高灵敏度LC-MS检测，为蛋白多肽类大分子的检测提供新的思路。

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