Development of an Automated On-line Hyphenation of Capillary Electrophoresis with Multicollector ICP-MS for Species-specific Isotopic Analysis of Biomolecules

到目前为止，采用MC-ICP-MS在线联用进行物种特异性同位素分析的研究很少，采用CE/MC-ICP-MS联用进行物种特异性同位素分析的研究也只有一篇。然而，这一领域在不同的研究领域都有很大的潜力。本项目将侧重于使用CE/MC-ICP-MS对元素物种进行同步同位素分析，特别是S，因为它是ICP-MS在生物系统中所有蛋白质中唯一的目标元素，也存在于肽和更小的重要生物分子中。S在生物分子中的同位素变化可用于阐明和追踪生物过程，以及帮助诊断疾病和监测治疗进展。此外，拟议的方法将促进生物研究中认证标准物质的发展，并支持该研究领域的计量活动。因此，本工作的主要目标是：PI) CE/MC-ICP-MS连字符的开发。自动化将尽可能多地实施，以便在没有操作员监督的情况下分析单个样品和序列。对于瞬态数据评估，将准备一个合适软件（MS Excel, Origin）中的模板，其中大多数评估过程将自动执行。为了验证目的，将制备合适的物种特异性二级参考标准品，并通过多收集器- icp - ms离线分离和分析，对硫iRM进行表征。然后将二级标准用于新开发的CE/MC-ICP-MS连字符的验证。由于同位素irm不仅非常昂贵，而且供应有限，因此它也可用作套标标准。PIII)将所开发的方法应用于ICP-ToF-MS，并随后比较CE/MC-ICP-MS和CE/ICP-ToF-MS在短瞬态信号中的同位素分析。PIV) CE/MC-ICP-MS和/或CE/ICP-ToF-MS方法在实际生物样品中生物分子的应用。首先，蛋氨酸、半胱氨酸和牛磺酸可以作为更简单的目标分子，然后发展到肽，如谷胱甘肽，小蛋白质，如金属硫蛋白，最后是大蛋白质，如人血清白蛋白。

Up to now, only a few studies have dealt with species-specific isotopic analysis by on-line hyphenation with MC-ICP-MS and only one with CE/MC-ICP-MS. However, this field holds great potential in varying research areas. This project will focus on the use of CE/MC-ICP-MS for the simultaneous isotopic analysis of elemental species, especially S, as it is the only target element for ICP-MS in all proteins in biological systems and is also present in peptides and smaller important biomolecules. Isotope variations of S in biomolecules could be used for the elucidation and tracing of biological processes, as well as help diagnosing diseases and monitoring treatment progress. Furthermore, the proposed method will foster the development of certified reference materials in bioresearch and support metrology activities in this research area.Therefore, the main objectives of this work are:PI) The development of a CE/MC-ICP-MS hyphenation. Automation will be implemented as much as possible, so that single samples and sequences can be analyzed without supervision of an operator. For transient data evaluation, a template in a suitable software (MS Excel, Origin) will be prepared, where most of the evaluation processes are executed automatically.PII) For validation purposes, a suitable species-specific secondary reference standard will be prepared and characterized against a sulfur iRM by off-line separation and analysis with multicollector-ICP-MS. The secondary standard will then be applied for the validation of the newly developed CE/MC-ICP-MS hyphenation. It may also be used as a bracketing standard since isotopic iRMs are not only very expensive but also of limited availability.PIII) Adaption of the developed method to ICP-ToF-MS and subsequent comparison of CE/MC-ICP-MS and CE/ICP-ToF-MS for isotopic analysis in short transient signals.PIV) The application of the developed CE/MC-ICP-MS and/or CE/ICP-ToF-MS method to biomolecules in real biological samples. For a start, methionine, cysteine and taurine can be used as more simple target molecules before advancing to peptides like glutathione, smaller proteins like metallothionein and finally larger proteins like human serum albumin.