Analysis of biological small molecule mixtures using multiple modes of mass spectrometric fragmentation coupled with new bioinformatics workflows

液相色谱-质谱（LC-MS）是化学、生物和生物医学科学中的一项重要技术。首先，小分子混合物被分离（LC），然后电离。之后，它们进入质谱，并根据它们的质量和离子的检测进行加速，随后分离。如果提供额外的能量，分子将分裂成离子，这是其结构的诊断。现代系统具有很高的分辨率，可以高精度地确定分子式。碎片可以在不同的能量水平上产生，它们的公式也可以确定。我们正在招标一套LC-MS系统，用于分析和鉴定复杂生物混合物中的小分子成分。小分子在生物学中具有重要的作用，如生物内部或生物之间的交流或与具有重要生物学效应的受体相互作用。生物学、生态学、生理学、生物化学、生物合成和人类疾病的基本问题只能通过研究这些复杂的分子混合物并了解它们的工作原理来回答。所提出的系统将能够高效快速地分离复杂的生物混合物，然后精确地确定每种组分的分子式。由于每个分子式都有许多可能的分子结构，因此具有不同能量水平的分子片段的能力对于增加分子鉴定的信心至关重要。应用包括小生物活性天然产物的发现和合成，对小分子生物合成的理解，对土壤中微生物过程的理解以及海绵和软体动物之间的化学交流，对疾病原因的理解以及药物发现。选择设备的参数是：在低分子量下的高质量精度；分离和破碎模式的范围和高动态范围，包括在强度上差异3-5个数量级的破碎峰的可能性。关键因素是能够准确、快速地识别丰度很低的化合物。我们选择了Thermo Scientific Orbitrap IQ-X混合质谱仪，该质谱仪具有四极杆分析仪，超高场轨道rap和离子阱分析仪，专门用于小分子分析。我们以前对该制造商的系统的经验是，它们具有非常高的动态范围，非常坚固，使用寿命长，并且具有出色的服务支持。为了证明该系统的实用性，我们选择了6个不同的项目：从深海和肠道细菌中发现新的生物活性化合物；发现可用于工业生物技术和合成生物学的新酶；环肽的发现、生物合成和自动分析在药物发现中具有重要意义；研究海洋无脊椎动物和大麦的代谢变化；了解修饰肽在人类疾病病因中的作用；传统药物在考古文物中的应用分析。新的MS还将使我们能够培训下一代科学家使用最先进的MS，这将使他们获得实践经验，这对他们未来的职业发展非常有益。我们以研究为主导的教学意味着即使是本科生也可以从最先进的设施中受益。拟议的仪器将被纳入，并将补充和显著提高我们现有的MS设施，通过增加新的功能。新的LC-MS不仅将使我们在阿伯丁的学生和员工受益，而且我们的学术和工业合作伙伴也将从接受培训和获得最先进的MS技术中受益。

Liquid chromatography-mass spectrometry (LC-MS) is an essential technique in the chemical, biological and biomedical sciences. To start with, a mixture of small molecules is separated (LC) followed by ionisation. After this, they enter the MS and are accelerated followed by separation according to their mass and detection of the ions. If additional energy is provided, the molecules will fragment into ions which are diagnostic of their structure. Modern systems have very high resolution to determine the molecular formulas with high accuracy. Fragments can be generated at different levels of energy and their formulas can also be determined.We are bidding for a LC-MS system to enable the profiling and identification of small molecule components in complex biological mixtures. Small molecules have important roles in biology such as communication within or between organisms or interacting with receptors with important biological effects. Fundamental questions in biology, ecology, physiology, biochemistry, biosynthesis and human disease can only be answered by studying these complex molecular mixtures and understanding how they work. The proposed system will enable efficient fast separation of complex biological mixtures followed by the accurate determination of the molecular formulas of each of the components. As there are many molecular structures possible for each molecular formula, the ability to fragment molecules with different levels of energy is essential to increase confidence in molecular identification. Applications include discovery and synthesis of small bioactive natural products, the understanding of biological synthesis of small molecules, understanding of microbial processes in soil and the chemical communication between sponges and molluscs, the understanding of disease causation as well as pharmaceutical discovery. Parameters for selecting the equipment are: high mass accuracy at low molecular weights; a range of separation and fragmentation modes and a high dynamic range including the possibility of fragmenting peaks differing in intensity by 3-5 orders of magnitude. The critical factor is the ability to accurately and quickly identify compounds present at very low abundance. We have selected the Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer which has a quadrupole analyser, ultra-high field orbitrap and an ion trap analyser and is promoted specifically for small molecule analysis. Our previous experience of systems from this manufacturer is that they have a very high dynamic range, are very robust with a long lifetime and have excellent service support.To demonstrate the utility of this system we have chosen 6 different projects: discovery of new bioactive compounds from deep-sea and gut bacteria; discovery of new enzymes that can be used in industrial biotechnology and synthetic biology; the discovery, biosynthesis and automated analysis of cyclic peptides which are becoming of major importance in drug discovery; to study variations in metabolism in marine invertebrates and barley; to understand the role of modified peptides in the causation of human disease; analysis of traditional medicine use in archaeological artefacts.The new MS will also enable us to train the next generation of scientists on the most advanced MS, which will allow them to gain hands-on experience that will be extremely beneficial for their future career development. Our research-led teaching means that even undergraduate students can benefit from the access to a state-of-the art facility. The proposed instrument will be incorporated into and will complement and significantly enhance our existing MS facility though the addition of new capabilities. The new LC-MS will not only benefit our students and staff in Aberdeen, but our academic and industrial collaborators will also benefit from receiving training and having access to the most advanced MS technologies.