NISA: Novel approaches for in situ analysis of biomolecules

这项研究的目的是开发直接从生物分子的自然(或实际)环境中分析生物分子，特别是蛋白质的新方法，即开发原位生物分子分析方法。蛋白质是细胞的马匹，执行生命所需的所有功能。它们还被用作治疗药物和消费产品。为了深入了解蛋白质在生命过程中的各种具体作用，或确定蛋白质药物的治疗效果，或确定消费产品中蛋白质添加剂的环境命运，有必要在分子水平上分析蛋白质。质谱学是根据电离分子的质量与电荷之比来表征电离分子的，它非常适合于这一挑战，提供了高灵敏度、广泛的特异性(所有分子都有质量)以及解释化学结构的能力。其最终目标是将分子分析直接与分子环境联系起来。就像法医负责确定非法物质的存在一样，与将样本移至单独地点和替代环境后进行的分析相比，在犯罪现场进行的分析具有更高的可靠性和可信度。越来越多的证据表明，原位蛋白质分析在生物标记物的发现、疾病的诊断和早期发现、靶向疗法(个性化药物)和治疗效果的评估方面具有开创性的作用。原位蛋白质分析的好处可以通过考虑药物治疗的肿瘤的薄组织切片来说明。原则上，原位分析将告知药物与目标的相互作用(即药物是否与正确的蛋白质结合？)。此外，通过原位蛋白质分析，消除了由于样品准备(例如，应用基质)或样品损坏而引入人工制品的能力。然而，仍然存在一些挑战。蛋白质是与大量化学修饰相关的大分子，它们与自身、其他蛋白质和其他分子类型形成松散结合的复合体。决定它们功能的不仅仅是它们的化学结构，还有它们的整体三维结构。在生物过程中极其重要的其他分子类也与蛋白质有着错综复杂的关系。任何原位质谱学方法都需要能够应对这些分析物驱动的挑战，即，它必须能够(A)测量蛋白质并表征任何修饰，(B)检测蛋白质复合体并确定其组成，(C)提供关于三维结构的信息，以及(D)检测其他相关的分子类别。此外，原位分析还面临着技术驱动的挑战，包括固有的高样品复杂性和广泛的浓度范围，以及定量的机会。研究将通过开发一种新的原位方法--液体萃取表面分析质谱仪，与两种互补类型的离子迁移率光谱相结合来应对这些挑战(它可以提供关于三维结构的信息，这项研究将主要在伯明翰大学生物科学学院的高级质谱学设施和化学学院的质谱学设施进行。该方案涉及许多学术和工业合作伙伴，在对国家物理实验室(NPL)、Thermo Fisher Science、Waters、Owlstone、佛罗里达州立大学、德克萨斯农工大学和马赛大学进行科学访问期间，将进行额外的研究。

The aim of the research is to develop novel approaches for the analysis of biomolecules, and in particular proteins, directly from their natural (or actual) environment, i.e., to develop approaches for in situ biomolecular analysis. Proteins are the work-horses of the cell and perform all the functions required for life. They also find uses as therapeutics and in consumer products. To gain insight into the various and specific roles of proteins in life processes, or to determine the therapeutic efficacy of protein drugs, or to establish the environmental fate of protein additives in consumer products, it is necessary to be able to analyse proteins at a molecular level. Mass spectrometry, in which ionised molecules are characterised according to their mass-to-charge, is ideally suited to this challenge, offering high sensitivity, broad specificity (all molecules have a mass), and the capability for chemical structure elucidation. The ultimate goal is to link molecular analysis directly to molecular environment. Much like a forensics officer tasked with determining the presence of an illicit substance, there is much greater reliability and credibility afforded to an analysis performed at the scene of the crime than to one performed following removal of the sample to a separate location and alternative surroundings.Growing evidence suggests in situ protein analysis has groundbreaking roles to play in biomarker discovery, diagnosis & early detection of disease, targeting therapeutics (personalised medicine) and assessment of therapeutic efficacy. The benefits of in situ protein analysis can be illustrated by considering a thin tissue section through a drug-treated tumour. In principle, in situ analysis would inform on drug-target interactions (i.e., is the drug binding to the correct protein?). Moreover, with in situ protein analysis the capacity for artefact introduction as a result of sample preparation (e.g., application of a matrix) or sample damage is eliminated.Nevertheless, a number of challenges exist. Proteins are large molecules associated with a vast array of chemical modifications, and which form loosely-bound complexes with themselves, other proteins and other molecule types. It is not only their chemical structure but also their overall 3-D structure which dictate their function. Other molecular classes that are hugely important in biological processes also have an intricate relationship with proteins. Any in situ mass spectrometry approach needs to be able to meet these analyte-driven challenges, i.e., it must be capable of (a) measuring proteins and characterising any modifications, (b) detecting protein complexes and determining their constituents, (c) providing information on 3-D structure, and (d) detecting other relevant molecular classes. Moreover, there are technique-driven challenges for in situ analysis including inherently high sample complexity and wide ranging concentrations, and opportunities for quantitation.The research will meet these challenges by developing a newly emerging in situ approach, liquid extraction surface analysis mass spectrometry, in combination with two complementary types of ion mobility spectrometry (which can either provide information on 3-D structure, or separate ionised molecules in the mass spectrometer on the basis of their 3-D shape) and a structural elucidation strategy known as electron-mediated dissociation mass spectrometry.The research will be undertaken primarily at the University of Birmingham in the Advanced Mass Spectrometry Facility in the School of Biosciences and the School of Chemistry mass spectrometry facility. The programme involves a number of academic and industrial collaborators and additional research will be carried out during scientific visits to National Physical Laboratory (NPL), Thermo Fisher Scientific, Waters, Owlstone, Florida State University, Texas A&M University and Université d'Aix-Marseille.