Novel Methods for the direct analysis of untreated biological samples using hyphenated ion mobility/ mass spectrometry with ambient ionisation

该项目是由阿斯利康研发中心Charnwood、临床药理学和DMPK开发新型分析技术的需求推动的。在现实世界的生物样品调查中所面临的技术挑战集中在对新方法的要求上，这些新方法结合了新兴技术的力量，以提供更高的灵敏度、选择性和样品吞吐量。这些生物应用领域侧重于测定生物标志物、肽和蛋白质片段、代谢物、蛋白质结合复合物和来自许多复杂样品基质的低分子量化合物，包括血浆、尿液和肺痰。传统的基于质谱的生物分子分析方法依赖于使用电喷雾（ESI）或基质辅助激光解吸/电离（MALDI）电离。虽然这些技术被广泛使用，但在分析之前都需要大量的样品制备。在生物样品中遇到的盐和其他内源性化合物存在时，ESI容易受到离子抑制，在LC-MS/MS分析之前，必须通过固相样品萃取去除这些化合物。MALDI对内源物质的耐受性较好，但需要在样品中加入基质。拟议的学生的目标是开发新的分析工具，允许使用解吸电喷雾电离（DESI）结合离子迁移谱法和质谱法（IM-MS）对生物分子进行直接，稳健的测量。DESI是一种新的电离方法，它允许生物分子直接和快速地从表面解吸，而无需事先对样品进行预处理。尽管与ESI相关，DESI技术已被证明对内源性物质的存在更为稳健。传统的质谱平台，就其本身的性质而言，在增加离子检测的选择性方面提供的很少，没有与采样DESI离子种群相关的重大数据损失，并且不可能通过色谱法分离解吸离子。然而，将DESI与离子迁移率（IM）光谱和质谱相结合，可以在质谱分析之前预先分离解吸离子。IM是一种气相电泳技术，它根据离子的电荷和碰撞截面（即大小和形状）在毫秒时间尺度上分离离子。混合离子迁移率质谱（IM-MS）的正交特性结合了电泳和质电荷分离，同时保留了desi衍生的离子数据。拉夫堡大学在许多基于离子迁移率的技术和环境样品电离方法方面拥有丰富的经验，包括DESI。项目主要成果如下：成果1：提供新颖、稳健的DESI-IM-MS，展示了对生物分子测定的高选择性和高灵敏度，以提高检测和确认。可交付2：将样品分析时间从数小时缩短到数秒，显著提高样品吞吐量并消除相关瓶颈。成果3：在使用DESI-IM-MS进行快速分析之前，通过对未经处理的样品（同位素稀释，内部标准化或稳定标签同位素）进行峰化，实现生物分子的快速定量。成果4：通过测量碰撞横截面对生物分子进行结构和构象分析，从而在更广泛的生物学背景下获得更好的理解，例如在给定生物系统中的蛋白质构象和功能。成果5：上述关键成果的结合将导致方法固有地转移到英国生物科学的重要科学平台，如代谢组学和蛋白质组学研究，其中复杂生物样品的敏感和选择性分析至关重要。

This project is driven by the need for development of novel analytical technology at AstraZeneca R&D Charnwood, Clinical Pharmacology and DMPK. The technological challenges faced in the investigation of real-world biological samples are centred on the requirement for new methods, which combine the power of emerging techniques to afford increased sensitivity, selectivity and sample throughput. These biological application areas focus upon the determination of biomarkers, peptide and protein fragments, metabolites, protein-bound complexes, and low-molecular weight compounds from a number of complex sample matrices, including plasma, urine, and lung sputum. Conventional mass spectrometry-based analytical methods for biomolecule analysis rely on the use of electrospray (ESI) or matrix-assisted laser desorption/ionization (MALDI) ionization. Whilst these techniques are widely used, both require extensive sample preparation prior to analysis. ESI is susceptible to ion suppression in the presence of salts and other endogenous compounds encountered in biological samples, which must be removed by solid-phase sample extraction prior to LC-MS/MS analysis. MALDI is more tolerant to endogenous material, but requires addition of matrix to the sample. The objective of the proposed studentship is to develop novel analytical tools that allow the direct, robust measurement of biomolecules using desorption electrospray ionization (DESI) combined with ion mobility spectrometry and mass spectrometry (IM-MS). DESI is a new approach to ionization that allows biomolecules to be desorbed directly and rapidly from surfaces without prior sample pre-treatment . Although related to ESI, the DESI technique has been shown to be more robust to the presence of endogenous material. Conventional mass spectrometric platforms, by their very nature, offer little in the way of increasing the selectivity of ion detection, without significant loss of data related to the sampled DESI ion population and separation of the desorbed ions by chromatography is not possible. However, combining DESI with ion mobility (IM) spectrometry and mass spectrometry allows the desorbed ions to be pre-separated prior to mass spectrometric analysis. IM is a gas-phase electrophoretic technique that separates ion on the basis of their charge and collision cross section (i.e. size and shape) on the millisecond timescale. The orthogonal nature of hybrid ion-mobility mass spectrometry (IM-MS) combines electrophoretic with mass-to-charge separation, whilst retaining the DESI-derived ion data. Loughborough University has extensive experience in a number of ion mobility-based techniques and ambient sample ionisation methods, including DESI. The key deliverables of this project are: Deliverable 1: The provision of novel and robust DESI-IM-MS, demonstrating high selectivity and sensitivity for the determination of biomolecules to improve detection and confirmation. Deliverable 2: Reducing sample analysis times from hours to seconds, dramatically increasing sample throughput and removing associated bottlenecks. Deliverable 3: Enable rapid quantitation of biomolecules by spiking untreated samples (isotope dilution, internal standardisation or stable-label isotopes) prior to rapid analysis using DESI-IM-MS. Deliverable 4: Structural and conformational analysis of biomolecules through measurement of collision cross section, allowing greater understanding in wider biological contexts, such as protein conformation and function in a given biological system. Deliverable 5: Combination of the key deliverables above would result in methods inherently transferable to important scientific platforms within the UK biosciences, such as metabolomic and proteomic research, where the sensitive and selective analysis of complex biological samples is paramount.