常压离子源在药物—血浆蛋白亲和力考察和游离血药浓度检测中的应用

发布时间：2018-06-17 13:09

本文选题：常压质谱技术 + 解吸附电喷雾质谱　；参考：《中国食品药品检定研究院》2014年硕士论文

【摘要】：近年来，随着药物-血浆蛋白亲和力以及游离血药浓度的研究领域的发展，研究者对其检测手段不断提出更高的要求，发展新的高通量、便捷的检测方法意义重大。常压质谱技术能直接、实时、快速、原位的提供分子的结构信息，将它应用于药物-血浆蛋白亲和力以及游离血药浓度的检测中，能为其研究提供一条新的途径。本论文着重研究了基于解吸附电喷雾离子源和文丘里简易常压超声喷雾离子源检测药物-血浆蛋白亲和力以及游离血药浓度的新方法。主要包括以下工作：一、利用解吸附电喷雾质谱（Desorption Electrospray Ionization MassSpectrometry, DESI-MS）解决了目前药物-血浆蛋白研究存在的三方面的问题。第一，在负电区域抑制解离机理的基础上，有负电位点的蛋白不会释放特异性结合的配体，本身可以作为参比蛋白，省去了为每一种药物寻找参比蛋白的过程。第二，提取离子流图检测的是药物分子的准分子离子峰，而不是药物-蛋白复合物，这样，气相库伦爆炸诱导解离可以起到一个积极的作用，而不是影响检测的作用。第三，我们自行搭建的全自动移动平台可以解决分析速度慢的问题。我们选择仅有一个特异性结合位点的α1-酸性糖蛋白作为模式蛋白，先前文献已经报导了DMOA可以作为α1-酸性糖蛋白的置换剂。我们的工作分为三部分，第一，用（R）、（S）型氨氯地平作为模式配体，证明了负电区域抑制解离机理，它们与α1-酸性糖蛋白的立体选择性特异性结合已经被报导。第二，我们用5种α-肾上腺素受体阻滞剂和10种β-肾上腺素受体阻滞剂证明了我们方法的高通量属性。我们在2.3分钟内获得了15*3个样本点的全部数据信息。第三，几乎不与α1-酸性糖蛋白发生作用的阿替洛尔和莫索尼定被选为内标物，以校正喷入离子传输管体积的差别，与先前的实验结果相一致，证明我们的方法至少是半定量的方法。二、利用文丘里-简易常压超声喷雾质谱（Venturi EasyAmbient Sonic-SprayIonization Mass Spectrometry, V-EASI-MS）建立了一种实时在线监测药物-负电位点蛋白结合的方法，体系由两部分组成：商业化的线性离子阱（LTQ）质量分析器和自行搭建的文丘里-简易常压超声喷雾离子源。我们的实验结果进一步证明了我们首先提出并验证的负电区域抑制解离机理（Anionic region inhibiteddissociation mechanism）。机理描述了负电区域在特异性结合配体解离中的作用。为了区分特异性以及非特异性的相互作用，，我们首先在没有特异性结合药物解离的条件下评估了非特异性的相互作用。所以我们优化了解吸附电喷雾直接分析的质谱条件，避免特异性结合药物解离。我们主要采用了两个策略来防止特异性结合药物从复合物上解离：第一，我们选择负电位点蛋白——α1-酸性糖蛋白，作为模式蛋白。使用负电位点蛋白获得质谱信号强度与亲和力的相关性比使用正电位点蛋白更好。第二，使用的文丘里-简易常压超声喷雾离子源是一种最软电离源，有利于保护对照试验中的特异性相互作用。
[Abstract]:In recent years, with the development of the research field of drug plasma protein affinity and free blood concentration, researchers have constantly put forward higher requirements for their detection methods, and the development of new high throughput and convenient detection methods is of great significance. The atmospheric mass spectrometry technology can provide the structural information of molecules in real time, fast speed and in situ, and apply it to the system. A new approach for the detection of drug plasma protein affinity and free blood concentration can be provided for its research. This paper focuses on the study of new methods for detecting the affinity of plasma protein and the concentration of free blood drugs based on desorption ion source and Venturi simple atmospheric pressure ultrasonic spray ion source. Make:
First, Desorption Electrospray Ionization MassSpectrometry (DESI-MS) is used to solve the three problems in the current drug plasma protein study. First, on the basis of the mechanism of the negative region inhibition, the protein with negative potential will not release the specific binding ligand itself, which itself can be used as a reference. Specific protein, it saves the process of searching for the reference protein for every drug. Second, the extraction ion flow graph is the excimer ion peak of the drug molecule, not the drug protein complex, so that the gas phase Kulun explosion induced dissociation can play a positive role, not the effect of detection. Third, we build it by ourselves. The full automatic mobile platform can solve the problem of slow analysis. We choose only one specific binding site of alpha 1- acid glycoprotein as a pattern protein. Previous literature has reported that DMOA can be used as a replacement agent for alpha 1- acid glycoprotein. Our work is divided into three parts. First, (R), (S) type amlodipine as a pattern match. The body, which proved the mechanism of the negative region inhibition of dissociation, and their stereoselective binding with alpha 1- acid glycoprotein has been reported. Second, we demonstrated our method with 5 alpha adrenergic receptor blockers and 10 beta adrenergic receptor blockers. We obtained 15*3 sample points within 2.3 minutes. All data information. Third, atenolol and mol SONY, which almost do not interact with alpha 1- acid glycoproteins, were selected as the internal standard to correct the difference in the volume of the injected ion transfer tube. It was consistent with the previous experimental results, proving that our method is at least a semi quantitative method.
Two, a method of real-time online monitoring of drug negative potential protein binding is established by using Venturi simple atmospheric pressure Sonic-SprayIonization Mass Spectrometry (V-EASI-MS). The system consists of two parts: commercial linear off Zi Jing (LTQ) quality analyzer and self built Venturi - Our experimental results further demonstrate the mechanism of Anionic region inhibiteddissociation mechanism which we first proposed and verified. The mechanism describes the role of the negative region in the dissociation of specific binding ligands in order to distinguish specific and nonspecific phases. Interaction, we first evaluated the nonspecific interaction without specific combination of drug dissociation. Therefore, we optimized the mass spectrum conditions of the direct analysis of the adsorption electrospray to avoid the specific binding of drugs. We mainly adopted two strategies to prevent the dissociation of the specific binding drugs from the complex: First, we choose the negative potential point protein, alpha 1- acid glycoprotein, as a pattern protein. The use of negative potential protein to obtain the correlation between the intensity and affinity of the mass spectrum is better than the positive potential point protein. Second, the Venturi simple atmospheric pressure ultrasonic spray ion source used is the most soft ionization source, which is beneficial to the protection of the control trial. Specific interaction.
【学位授予单位】：中国食品药品检定研究院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：O657.63;R927

【共引文献】