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

使用联用离子淌度/质谱法和环境电离直接分析未经处理的生物样品的新方法

• 批准号: BB/G017557/1
• 金额: $ 9.48万
• 依托单位: Loughborough University
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG017557%2F1
• 关键词: Novel; Methods; direct; analysis; untreated

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.

该项目是由阿斯利康R&D Charnwood，临床药理学和DMPK开发新型分析技术的需求驱动的。在调查真实世界生物样本方面面临的技术挑战主要集中在对新方法的需求上，这些新方法将新兴技术的力量联合收割机结合起来，以提高灵敏度、选择性和样本处理量。这些生物学应用领域集中于从许多复杂样品基质（包括血浆、尿液和肺痰）中测定生物标志物、肽和蛋白质片段、代谢物、蛋白质结合复合物和低分子量化合物。用于生物分子分析的常规的基于质谱的分析方法依赖于电喷雾（ESI）或基质辅助激光解吸/电离（MALDI）电离的使用。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。ESI在生物样品中遇到的盐和其他内源性化合物的存在下易受离子抑制，这些化合物必须在LC-MS/MS分析之前通过固相样品萃取除去。MALDI对内源性物质更耐受，但需要向样品中添加基质。拟议的学生奖学金的目的是开发新的分析工具，允许使用解吸电喷雾电离（DESI）结合离子迁移谱和质谱（IM-MS）直接，可靠地测量生物分子。DESI是一种新的电离方法，它允许生物分子直接快速地从表面解吸，而无需预先进行样品预处理。尽管与ESI相关，但DESI技术已被证明对内源性物质的存在更稳健。传统的质谱平台，就其本质而言，在增加离子检测的选择性方面提供很少，而没有与采样的DESI离子群相关的数据的显著损失，并且通过色谱法分离解吸的离子是不可能的。然而，将DESI与离子迁移率（IM）谱法和质谱法组合允许在质谱分析之前预先分离解吸的离子。IM是一种气相电泳技术，其在毫秒时间尺度上基于离子的电荷和碰撞横截面（即尺寸和形状）分离离子。混合离子迁移率质谱（IM-MS）的正交性质结合了电泳与质荷分离，同时保留了质谱衍生的离子数据。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。该项目的主要交付成果是：可实现1：提供新型和强大的生物质谱-离子迁移-质谱，证明了生物分子测定的高选择性和灵敏度，以改善检测和确认。优点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。可选项3：在使用EST-IM-MS进行快速分析之前，通过掺入未处理的样品（同位素稀释，内部标准化或稳定标记同位素）来实现生物分子的快速定量。表4：通过测量碰撞截面进行生物分子的结构和构象分析，从而更好地了解更广泛的生物背景，例如给定生物系统中的蛋白质构象和功能。可选项5：上述关键交付成果的结合将使方法本身可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，其中对复杂生物样品的敏感和选择性分析至关重要。