Shu Chang, Li Teng-Fei, Li Duo, Li Zhong-Qiu, Xia Xing-Hua
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
School of Pharmacy, Department of Clinical Pharmacology, Sir Run Hospital, Nanjing Medical University, Nanjing 211166, China.
Analyst. 2021 Mar 8;146(5):1725-1733. doi: 10.1039/d0an01948h.
Protein drugs showing strong pharmaceutical activity, high specificity, and low toxicity and side effects have drawn extensive attention in the field of life sciences and medicine. Precise evaluation of the function of these drugs requires accurate and sensitive detection methods. Here, we report a novel chromatography-tandem mass spectrometry (LC-MS/MS) method for sensitive and selective detection of protein drugs. Magnetic nanoparticles (Apt29@MNPs) were functionalized by thrombin aptamers, and quantum dots (Apt15@ss@QDs) were dual-functionalized with quantitative thrombin aptamers and small molecules with high ionization efficiency as the mass barcode. After Apt29@MNPs specifically purify and enrich thrombin from biological samples, they can form a nano "sandwich structure" when Apt15@ss@QDs are added, resulting in the release of the mass barcode for LC-MS/MS analysis via the cutting of the disulfide bond. Since there is a higher quantitative molecular ratio of mass barcode to thrombin in the nano-"sandwich structure", quantitative detection of thrombin with high sensitivity and selectivity can be achieved via the LC-MS/MS detection of the mass barcode with high ionization efficiency rather than thrombin, which effectively avoids the disadvantages of direct protein detection by mass spectrometry. The established method for thrombin detection shows a good linear relationship in a concentration range of 0.00115-1.15 nM with a limit of detection (LOD) of 0.0007 nM. The present work provides a new approach for the effective and sensitive quantitative analysis of protein drugs and would be of great significance in promoting the development of protein drugs and clinical applications.
具有强大药物活性、高特异性以及低毒性和副作用的蛋白质药物在生命科学和医学领域引起了广泛关注。对这些药物功能的精确评估需要准确且灵敏的检测方法。在此，我们报道了一种用于灵敏且选择性检测蛋白质药物的新型色谱 - 串联质谱（LC - MS/MS）方法。磁性纳米颗粒（Apt29@MNPs）用凝血酶适配体进行功能化修饰，量子点（Apt15@ss@QDs）用定量凝血酶适配体和具有高电离效率的小分子作为质量条形码进行双功能化修饰。在Apt29@MNPs从生物样品中特异性纯化和富集凝血酶后，当加入Apt15@ss@QDs时，它们可形成纳米“三明治结构 ”，通过二硫键的切割导致质量条形码释放用于LC - MS/MS分析。由于在纳米“三明治结构”中质量条形码与凝血酶的定量分子比更高，因此可通过对具有高电离效率的质量条形码而非凝血酶进行LC - MS/MS检测来实现对凝血酶的高灵敏度和高选择性定量检测，这有效避免了直接通过质谱检测蛋白质的缺点。所建立的凝血酶检测方法在0.00115 - 1.15 nM浓度范围内呈现良好的线性关系，检测限（LOD）为0.0007 nM。本工作为蛋白质药物的有效且灵敏的定量分析提供了一种新方法，对推动蛋白质药物的开发和临床应用具有重要意义。
