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.

中文翻译：

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构建用于用户指定目标蛋白多重定量的点菜 QconCAT 蛋白质标准品

QconCAT 是用于蛋白质组学应用的定量串联体，可产生化学计量数量的稳定同位素标记内标集。然而，例如，改变 QconCAT 设计以替换性能不佳的肽标准是一个漫长的过程。我们报告了一种组装和构建 QconCAT 的新方法，基于生物砖的合成生物学规则，利用循环组装从单个生物砖构建更大的实体。基本构建块 (Qbrick) 是一段 DNA，它为单个蛋白质编码两个或多个定量肽，可作为文库资源轻松保存在存储库中。然后将这些 Qbrick 组装在单管连接反应中，强制执行组装顺序，以产生可用于小量化产品的短 QconCAT。然而，短构建体的 DNA 环境还允许进行第二个环组装循环，这样五个不同的短 QconCAT 可以在第二次单管连接中组装成更长的 QconCAT。从 Qbricks 库中，可以快速有效地以适合体内或体外表达和标记的形式组装定制的 QconCAT。我们将这种方法称为 ALACAT 策略，因为它允许量化标准的点菜设计。ALACAT 方法是 QconCAT 实施灵活性的主要增益，因为它支持 QconCAT 的快速编辑和改进，并允许例如用另一种肽替换一种肽。从 Qbricks 库中，可以快速有效地以适合体内或体外表达和标记的形式组装定制的 QconCAT。我们将这种方法称为 ALACAT 策略，因为它允许量化标准的点菜设计。ALACAT 方法是 QconCAT 实施灵活性的主要增益，因为它支持 QconCAT 的快速编辑和改进，并允许例如用另一种肽替换一种肽。从 Qbricks 库中，可以快速有效地以适合体内或体外表达和标记的形式组装定制的 QconCAT。我们将这种方法称为 ALACAT 策略，因为它允许量化标准的点菜设计。ALACAT 方法是 QconCAT 实施灵活性的主要增益，因为它支持 QconCAT 的快速编辑和改进，并允许例如用另一种肽替换一种肽。