Johnson, James, Harman, Victoria M 
ORCID: 0000-0002-0990-153X, Franco, Catarina, Emmott, Edward ORCID: 0000-0002-3239-8178, Rockliffe, Nichola, Sun, Yaqi, Liu, Lu-Ning ORCID: 0000-0002-8884-4819, Takemori, Ayako, Takemori, Nobuaki and Beynon, Robert J ORCID: 0000-0003-0857-495X
(2021)
Construction of a la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.
BMC BIOLOGY, 19 (1).
195-.
Abstract
<h4>Background</h4>QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process.<h4>Results</h4>We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. 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 construct also allows a second cycle of loop 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.<h4>Conclusions</h4>We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.
| Item Type: | Article |
|---|---|
| Additional Information: | QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. |
| Uncontrolled Keywords: | Synthetic biology, Standards, Absolute quantification, Stable isotopes, QconCAT, Quantitative proteomics, Loop assembly |
| Divisions: | Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology Faculty of Health and Life Sciences > Tech, Infrastructure and Environmental Directorate |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 22 Sep 2021 15:04 |
| Last Modified: | 15 Dec 2022 07:10 |
| DOI: | 10.1186/s12915-021-01135-9 |
| Open Access URL: | https://bmcbiol.biomedcentral.com/articles/10.1186... |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3137929 |
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