Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.
Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.
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DOI：
10.1186/s12915-021-01135-9
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发表时间：
2021-09-08
期刊：
影响因子：
5.4
通讯作者：
Beynon RJ
中科院分区：
文献类型：
--
作者：
Johnson J;Harman VM;Franco C;Emmott E;Rockliffe N;Sun Y;Liu LN;Takemori A;Takemori N;Beynon RJ
qconcat是用于蛋白质组学应用的定量concatamers，可产生化学计量数量的稳定同位素标记的内部标准。然而，改变QconCAT设计，例如，取代表现不佳的肽标准是一个漫长的过程。基于生物砖的合成生物学原理，我们提出了一种组装和构建qconcat的新方法，利用环路组装从单个生物砖构建更大的实体。基本构建块（Qbrick）是为单个蛋白质编码两个或多个定量肽的DNA片段，易于作为库资源保存在存储库中。然后，这些qbrick在单管连接反应中组装，强制组装顺序，以产生可用于小量化产品的短qconcat。然而，短结构的DNA环境也允许环组装的第二个周期，这样五个不同的短QconCAT可以在第二次单管连接中组装成一个更长的QconCAT。从Qbricks库中，定制的QconCAT可以快速有效地组装成适合体内或体外表达和标记的形式。我们将这种方法称为ALACAT策略，因为它允许任意设计量化标准。ALACAT方法是QconCAT实现灵活性的主要增益，因为它支持快速编辑和改进QconCAT，并允许，例如，用另一个肽替代一个肽。在线版本包含补充材料，可在10.1186/s12915-021-01135-9获得。
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. 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. 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. The online version contains supplementary material available at 10.1186/s12915-021-01135-9.
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影响因子：
4.8
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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