Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification
Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification
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合成生物学遇上蛋白质组学：构建用于绝对蛋白质定量的点菜 QconCAT
DOI：
10.1101/2021.04.13.439592
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Johnson J
中科院分区：
文献类型：
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作者：
Johnson J
我们报告了一种组装和构建 QconCAT 的新方法，QconCAT 是用于蛋白质组学应用的定量连接体，可产生化学计量数量的稳定同位素标记的内标组。新方法基于生物砖的合成生物学原理，利用循环组装从单个生物砖构建更大的实体。它极大地提高了 QconCAT 实施的灵活性，并实现了快速高效的可编辑性，例如可以用一种肽替换另一种肽。基本构建块（Qbrick）是一段 DNA，为单个蛋白质编码两个或多个定量肽，可轻松作为库资源保存在存储库中。然后将这些 Qbrick 在单管连接反应中组装，强制执行组装顺序，以产生可用于小定量产品的短 QconCAT。然而，短的 DNA 背景还允许第二个组装周期，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，因为它允许按菜单设计量化标准。
We report a new approach to the assembly and construction of QconCATs, quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. The new approach is based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. It offers a major gain in flexibility of QconCAT implementation and enables rapid and efficient editability that permits, for example, substitution of one peptide for another. 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 also allows a second cycle of 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 vivoorin vitro. We refer to this approach as the ALACAT strategy as it permitsà la cartedesign of quantification standards.