Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification



Johnson, James ORCID: 0000-0002-8849-0993, Harman, Victoria, de Matos Ferraz Franco, Catarina, Rockliffe, Nicola, Sun, Yaqi ORCID: 0000-0002-2175-7905, Liu, Luning, Takemori, Ayako, Takemori, Nobuaki and Beynon, Robert
(2021) Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification.

Access the full-text of this item by clicking on the Open Access link.
[img] Text
ALACATS_030521ref.pdf - Submitted version

Download (14MB) | Preview

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: Article
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: 05 Oct 2022 20:44
DOI: 10.1101/2021.04.13.439592
Open Access URL: https://www.biorxiv.org/content/10.1101/2021.04.13...
URI: https://livrepository.liverpool.ac.uk/id/eprint/3132125