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

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.

Since the commercial introduction of soft ionisation techniques, mass spectrometry has become one of the most important research tools not only for chemists, but also for a wide range of other research fields including environmental research and biomedicine. We propose to buy a Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer which has quadrupole analyser, ultra-high field orbitrap and an ion trap analyser and is promoted specifically for small molecule analysis. It has 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. This system can obtain accurate masses of fragments up to MS10 and has a maximum resolution of 500,000 at lower scan rates. These specifications mean we can obtain deep ion trees on minor components in complex biological mixtures so they can be identified reliably.
This system will complement our existing MS facilities, providing new capabilities to significantly enhance research outcomes in several UKRI funded projects. The new instrument will be incorporated into our existing MS facility in the Department of Chemistry. Once it is fully operational, the technician and superusers will receive training from the vendor on method development, operation and maintenance of the new instrument. Full training will be provided to all future users by the technician and superusers. Instrument access will be managed using our existing booking system, and trained users can book and use the instrument 24/7. Project prioritisation and future research development will be managed by academics and overseen by the management committee. Through Aberdeen Core Facilities Services and existing academic and industrial collaborations, external users can receive access to the new facility.