ALACATS: bespoke strategy for absolute protein quantification
Lead Research Organisation:
University of Manchester
Department Name: School of Biological Sciences
Abstract
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Technical Summary
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.
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.
Planned Impact
The proposal is directly relevant to BBSRC priorities: "Basic bioscience underpinning health", "Data driven biology", "Systems approaches to the biosciences", "Healthy ageing" and "Technology development for the biosciences".
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, as well as through the Rosalind Franklin Institute.
Training
The researchers supported by this proposal will develop high level skills in mass spectrometry and data processing workflows, in the area of research software engineering in particular through interactions with the University central Research IT team at Manchester and the Software Sustainability Insitute. 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.
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, as well as through the Rosalind Franklin Institute.
Training
The researchers supported by this proposal will develop high level skills in mass spectrometry and data processing workflows, in the area of research software engineering in particular through interactions with the University central Research IT team at Manchester and the Software Sustainability Insitute. 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.
Publications
Rusilowicz M
(2023)
AlacatDesigner-Computational Design of Peptide Concatamers for Protein Quantitation.
in Journal of proteome research
Creamer DR
(2024)
Isoform-specific sequestration of protein kinase A fine-tunes intracellular signaling during heat stress.
in Cell reports
Description | We designed a software tool for selecting peptides from proteins which are excellent surrogate markers for these parent proteins, and made this available. The associated publications shows how this can be used with some examples cases, and provides guidelines for design and use. |
Exploitation Route | The software and approach are in active use at Manchester and other labs, and we have factored them into several further grant applications, currently under review. It has also proven very effective on BBSRC DTP studentship allowing us to quantify stoichiometries of protein kinase A subunits in yeast and we hope to publish this in the next 12 months |
Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Early days, but we have helped design peptide standards that could be used in measuring allergen levels in foodstuffs which in turn might influence national standards agencies. Currently this work has not yet been taken forwards, though we have successfull design peptide standards in ALACATs for yeast protein samples. |
First Year Of Impact | 2022 |
Sector | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology |
Impact Types | Policy & public services |
Description | Rosalind Franklin Institute |
Organisation | Rosalind Franklin Institute |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | We are working with the RFI to help design protein standards for their members who wish to absolutely quantify proteins in the proteome. |
Collaborator Contribution | We plan to hold workshops and meetings to showcase the technology we are developing. The RFI have contributed financially to this work so the cost to users will be negligible. We have not hosted any yet but they are being planned. |
Impact | to come |
Start Year | 2020 |
Title | AlaCat Designer |
Description | The Alacat designer supports users in the selection of quantotypic peptides for use in protein quantificaiton via external standards that are spiked in to a mixture. We first presented this at BPSR 2021 in a virtual conference |
Type Of Technology | Software |
Year Produced | 2021 |
Impact | Some interest from commerical parties who sell recomibant protein products but nothing materialsed of yet |
URL | https://monod.ls.manchester.ac.uk/alacat/ |