Construction of \xe0 la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins

James Johnson,Nobuaki Takemori,Catarina Franco,Robert J Beynon,Victoria M Harman,Edward Emmott,Nichola Rockliffe,Yaqi Sun,Lu-Ning Liu,Ayako Takemori

doi:10.1186/s12915-021-01135-9

James Johnson, Nobuaki Takemori + Show 8 more

Open Access

https://doi.org/10.1186/s12915-021-01135-9

Copy DOI

Journal: BMC biology	Publication Date: Sep 8, 2021
Citations: 8	License type: open-access

Affiliation: University of Liverpool, Ehime University

Abstract

BackgroundQconCATs 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.ResultsWe 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.ConclusionsWe 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.

Highlights

QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards
Absolute quantification of proteins by mass spectrometry is typically based on the use of accurately quantified stable isotope labelled internal standards, usually peptides, as surrogates for the protein quantification
QconCATs are multiplexed protein standards for proteomics, products of artificial genes designed to encode concatamers of peptides, wherein each peptide or more commonly, a pair of peptides, is chosen to act as mass spectrometric standard(s) for absolute quantification of multiple peptides

Summary

Introduction

QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. Changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. Absolute quantification of proteins by mass spectrometry is typically based on the use of accurately quantified stable isotope labelled internal standards, usually peptides, as surrogates for the protein quantification. An additional approach is the use of QconCAT technology [4, 8, 9]. QconCATs are multiplexed protein standards for proteomics, products of artificial genes designed to encode concatamers of peptides, wherein each peptide or more commonly, a pair of peptides, is chosen to act as mass spectrometric standard(s) for absolute quantification of multiple peptides. The initial publications on QconCATs [10,11,12] have received over 1000 citations and the methodology is well known and embedded in the community.

Methods

Results

Discussion

Conclusion