Johnson, James 
ORCID: 0000-0002-8849-0993, Harman, Victoria ORCID: 0000-0002-0990-153X, de Matos Ferraz Franco, Catarina, Rockliffe, Nicola, Sun, Yaqi ORCID: 0000-0002-2175-7905, Liu, Luning ORCID: 0000-0002-8884-4819, Takemori, Ayako, Takemori, Nobuaki and Beynon, Robert ORCID: 0000-0003-0857-495X
(2021)
Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification.
[Preprint]
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Abstract
We report a new approach to the assembly and construction of QconCATs, quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. The new approach is based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. It offers a major gain in flexibility of QconCAT implementation and enables rapid and efficient editability that permits, for example, substitution of one peptide for another. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short also allows a second cycle of assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. We refer to this approach as the ALACAT strategy as it permits a la carte design of quantification standards.
| Item Type: | Preprint |
|---|---|
| Uncontrolled Keywords: | 3101 Biochemistry and Cell Biology, 31 Biological Sciences, Biotechnology, Generic health relevance |
| Divisions: | Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 02 Aug 2021 07:35 |
| Last Modified: | 07 Dec 2024 21:19 |
| DOI: | 10.1101/2021.04.13.439592 |
| Open Access URL: | https://www.biorxiv.org/content/10.1101/2021.04.13... |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3132125 |
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