RapidFire Enhancement of Mass Spectrometry Capability Across the Biosciences

A major current challenge in biological science is to capitalise on the wealth of genomic information arising from DNA sequencing, to characterise the function and structure of DNA-encoded proteins. However, while genome sequencing has become very rapid, no methods currently exist which can experimentally determine the molecular details of the gene-products on a comparable timescale. As such the gulf between our understanding of what proteins are present in an organism and how they orchestrate the cellular processes necessary to life is ever increasing. Mass spectrometry (MS) is a method that has revolutionised the experimental identification of proteins and quantification of their cellular abundances. In Oxford we have pioneered a number of MS-based approaches to determine the structures, interactions, and dynamics of proteins, and to elucidate their biochemical roles in cells and whole organisms. However, we now need technology that can help analyse and exploit function at a rate comparable to that of data generated by the genomic revolution. As a step towards this, we propose to acquire a state-of-the-art RapidFire MS system to enable a step-change in the rate of our ability to accurately identify, analyse and quantify function in biological processes.

Specifically, the RapidFire 365 coupled to a high resolution iFunnel6550 QTof MS, will enable multiple current and future BBSRC researchers to dramatically increase the throughput at which we can screen biological reactions by over two orders of magnitude, including the analysis of biological reactions in real time. The dramatic increase in speed will revolutionise our ability to obtain functional assignments and identify ways to manipulate biological function on a biochemical level.

The new instrumentation will be integrated into dedicated laboratories in the Department of Chemistry in Oxford, providing new capabilities within the context of a carefully selected and comprehensive collection of instruments aimed at applying MS to the frontiers of chemical biology. The RapidFire MS system will be genuinely multi-user, enabling BBSRC-funded research by a large number of research groups in Oxford and the UK, through well-defined access routes overseen by experienced staff. The instruments will support research projects spanning a range of BBSRC strategic research priority areas, and will become an integrated piece of equipment in the Interdisciplinary Biosciences Doctoral Training Programme, thus making it readily available to researchers at other sites within this programme including Oxford Brookes University and the Diamond Light Source.

A major challenge in biological science is determining the physiologically relevant functions of gene products, both proteins and nucleic acids. Genetic methods are powerful, but biochemical investigations are essential to rationally assign and exploit the function of gene products. In particular, understanding enzymatic mechanisms of action, assaying their activity quantitatively, and identifying/probing ways to manipulate their activity on a reasonable timescale remains extremely difficult. It is currently a huge challenge for this kind of functional investigation to keep up with the information provided by the genomic revolution.

We will address this challenge by capitalizing on recent innovations in biological MS; very high throughput MS screening is emerging as one of the best means for functional assignments. We propose to acquire an MS system that will allow robust and high-throughput screening of ligands, inhibitors and products of catalysis. The RapidFire 365 is a high-throughput MS system incorporating cutting-edge micro-fluidics to rapidly inject samples, achieving <10 s sample-cycle time. Over 60 384-well plates can be loaded onto the system and automated analysis for up to 60 hrs initiated. The iFunnel 6550 QTof MS system incorporates a state-of-the-art LC system with liquid handling robotics to allow rapid analysis of samples, which can then be analysed by automated software for rapid determination of outcome.

This instruments, various iterations of which we have extensively road tested for over a decade, will revolutionize our ability to perform functional studies on proteins ranging from monomeric enzymes to multicomponent molecular machines across the diverse spectrum of BBSRC research, ranging from artificial RNA synthesis to antibiotic resistance and monitoring plant-pathogen interactions, and provide a resource for BBSRC-funded researchers to undertake experiments not currently possible in this country.

This new equipment represents a novel approach aimed at addressing an important problem, namely the multi-level characterisation of gene products. The RapidFire will complement our existing MS equipment for proteomics and structural and chemical biology, and will have significant impact on our research by providing a high throughput capability for the first time which will be used for elucidating the mechanisms underpinning enzyme function. The equipment will be truly multi-user, enabling research across the BBSRC remit, and will allow us to place Oxford at the forefront of international biological MS. We are committed to ensuring that the publicly funded equipment will play an important role in contributing to translating fundamental research into positive benefit for the health and economic prosperity of UK society.

Basic Science:
The research enabled by the new technologies is likely to have significant downstream impact far beyond the already large community of scientists working on the structure and function of proteins. The research areas of the many users of the proposed equipment span BBSRC Strategic Research Priorities (see Case for Support). Based on the track-record of the applicants, the research enabled will be published in high-quality journals, presented at international conferences, and if appropriate, filed as patent applications or used to form spin out companies, with which Oxford has an outstanding track record (e.g. £16.9m for OxStem and its subsiduaries: http://www.oxstem.co.uk/).

Industry:
The pharmaceutical, agrochemical, and biotechnology industries are vital to the UK economy, employing >250,000 people and generating billions of pounds of tax-revenue. Reliable and high-throughput screening methods underpin much of the research in these industries. There are already a few instances of RapidFire instruments in such companies, and while there is no additional capacity for our research activities, we will actively engage these companies (e.g. UCB) to share our methodologies. We have extensive links with industry for collaborative research including with GSK, Syngenta, GlycoVaxyn AG, ATDBio and AstraZeneca.

Training:
The multi-user equipment will be used to directly train >70 PhD students per year in biological MS technologies as part of their introductory year on BBSRC-funded DTC and DTP PhDs (http://www.biodtp.ox.ac.uk/) as well as the EPSRC-funded Synthesis for Biology and Medicine CDT (http://www.oxfordsynthesiscdt.ox.ac.uk/). In addition, >300 full-time researchers in Oxford will have access to this instrumentation and its capability, with the opportunity to be trained in its operation, and the analysis of the ensuing data for those studying non-human proteins in particular. Similarly, visitors from laboratories outside Oxford will potentially benefit from exposure to these techniques when accessing the instruments.

General public and related outreach:
The University has established routes for engaging the general public with our research. Aside from the engagement activities on-going in the labs of all the users, we also regularly host summer students give tours of the MS facility (including demonstrations) to school children; and invite prospective University applicants to see the labs during Open Days, so they can experience a cutting-edge research environment. Recent outreach activities include museum exhibitions focusing on BBSRC-related work including 'Biosense' and 'Back from the Dead', the first focusing on how basic BBSRC science led to the discovery of a family of oxygen sensors and the latter on antibiotic discovery and resistance (see http://www.mhs.ox.ac.uk/backfromthedead/).