ALACATS: bespoke solutions for absolute protein quantification

A famous biochemist, Arthur Kornberg, won the Nobel Prize for his work on the mechanisms by which DNA copies itself from cell to cell, generation to generation. But, he was acutely aware that whilst DNA (and by association, RNA) are the blueprint, the true vocation of life lies in the actions of the machines that are described in the nucleic acid blueprint, the proteins. To truly understand living processes, we need to gain a detailed quantitative understanding of the protein world. And, just as the field of genomics has transformed our knowledge of DNA, so an equivalent field of 'proteomics' has hugely advanced our understanding of the protein world.

The core technology in proteomics is based on sophisticated mass spectrometers, capable of analysing one million millionths of a gram of peptide in exquisite detail (we use peptides as the proxy molecule for their parent proteins). But sophisticated as they are, mass spectrometers all have one intrinsic limitation - they give different signal intensities for different peptides from the same protein, even though they are in the same amount. Yet, to understand how a cell is the manifestation of the proteins it contains, we need to be able to measure exactly how many copies of any one protein there are. To overcome this limitation, we use accurately known standards that are co-analysed by the mass spectrometer, with the advantage that the standard and true cellular protein can be separately measured because we engineer the standard to be 'heavier' and thus discernible in the mass spectrometer. Thus, if we add 1000 molecules of a standard, and the cell component gives us a signal that is twice as large, we can confidently assert that the sample contains 2,000 copies of that protein.

About 12 years ago, we invented a new method to generate large numbers of standards for quantitative proteomics. We created new 'designer proteins', never seen before on the planet, that could be made, in heavy form, by simple production in bacteria. These artificial proteins each contained peptide standards for up to 50 proteins.

Because these proteins were pre-designed in terms of the proteins that were encoded within it, it meant that they were not always perfectly tuned to the needs of individual scientists. What was needed was the ability to 'build your own' designer protein. In this proposal, we have devised a way to do exactly this. In future, no matter what the system of interest, scientists will be able to 'dial up' their interesting proteins, and we will be able to assemble, 'a la carte' a protein standard. We will create a library of building blocks (we call them 'Qbricks', short for 'Quantification biobricks') and using advanced synthetic biology methods of DNA manipulation we will be able to create, in two days, the perfect standard protein for their research. We call these 'ALACATs' because of the 'à la carte' design philosophy.

This is a revolutionary approach to absolute quantitative proteomics, and has huge potential to enhance our understanding of the protein world. This project will establish the core technology and methodologies, and build a set of Qbricks that will be used to create standards and research tools for the proteomics community. We will show how the ALACAT philosophy can be developed as a technical resource, readily drawn upon by many research groups, and thus, enabling a broad series of research programmes in a sustainable fashion.

QconCATs are artificial designer proteins that are used as internal standards for mass spectrometry-based quantification of proteins. By incorporating peptides from multiple proteins into a single construct we maintain stoichiometry, achieve high levels of labelling and permit multiple proteins to be quantified simultaneously and, importantly, in absolute terms i.e. copies per cell. QconCATs are designed by users with specific interests, and as such the proteins encoded in each QconCAT might not be optimal for other users.

We now propose a radical evolution of the QconCAT approach, and wish to develop a system whereby a QconCAT can be assembled to order, according to a list of target proteins specified by the user. We have conceived a novel approach to à la carte assembly of peptides reporting on specific target proteins into QconCATs using the precepts of synthetic biology. We call these constructs ALACATS to reflect the a la carte design of concatamers. We will build ALACATS from Qbricks, short double stranded oligonucleotides, typically 150bp. Individual Qbricks are then assembled into a two-stage process, building short or long ALACATs using a new approach called 'loop assembly'. The 'one Qbrick, one protein' approach is a major step change, and we foresee a growing library of Qbricks for a broad range of applications.

We seek support to develop the software to optimise Qbricks selection and design, the synthetic biology strategy and infrastructure to design, synthesise, validate and distribute ALACATS to the community. The set of standards will be targeted in collaboration with the Rosalind Franklin Institute, and built to the highest standards to support the most accurate, absolute quantification pipelines. By creating a totally flexible and user-focused design workflow, we will extend the reach, speed of deployment and accessibility of absolute quantification.

Economic and Societal impact
The novel approach we will deliver will create wholly new data sets that will drive insight into a diverse range of subjects. Academic research laboratories, biotechnology companies and pharmaceutical companies will benefit from this research. Similarly, this will also provide an excellent showcase for one of BBSRC's synthetic biology Foundries. We will ensure wide advocacy of the approach through scientific channels and publications.

Training
The researchers supported by this proposal will develop high level skills in mass spectrometry and data processing workflows. They will gain experience in writing papers and presenting their work at international meetings. Additionally, both partners will commit to presenting a further instance of our highly regarded quantitative proteomics course in collaboration with the Biochemical Society. The RFI Stakeholder meetings will also provide a forum for training and feedback on the webtools and interface. GeneMill will co-host an international training workshop in 2020 on synthetic biology.

Outreach
Project staff are active in in open days, in hosting visitors and in visiting school and public venues to talk about the ideas and delivery of advanced biological science. We have an impressive record of outreach activities, having taken part in Cafés Scientifiques, 'Meet the Scientists' at the World Museum, the Genetics Roadshow and Summer Science Clubs. We have built strong links with local colleges, bringing students to interview scientists, running MS samples and learning gel separations to be run at college. We will continue these programmes.