論文

査読有り 国際誌
2021年9月8日

Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.

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

19
1
開始ページ
195
終了ページ
195
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1186/s12915-021-01135-9

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

リンク情報
DOI
https://doi.org/10.1186/s12915-021-01135-9
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/34496840
PubMed Central
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425055
ID情報
  • DOI : 10.1186/s12915-021-01135-9
  • PubMed ID : 34496840
  • PubMed Central 記事ID : PMC8425055

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