Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
Anal Bioanal Chem. 2012 Sep;404(4):977-89. doi: 10.1007/s00216-012-6230-1. Epub 2012 Jul 9.
Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.
系统生物学需要了解蛋白质的绝对数量，以便对生物过程进行建模和模拟特定模型参数变化的影响。定量连接物（QconCAT）作为一种方法被建立起来，为同位素稀释标准实验中的一组目标蛋白质提供了多重绝对肽标准。将两个或更多个代表每个目标蛋白质的定量肽连接到一个设计基因中，该基因在大肠杆菌或其他细胞或无细胞系统中用稳定同位素进行代谢标记。用已知量的 QconCAT 与目标蛋白质共消化，生成一组标记的参考肽标准品，用于未标记的分析物对应物，并且通过使用适当的质谱平台，比较肽比的强度可以提供编码肽的绝对定量，进而提供它们所代表的目标蛋白质的定量。在这篇综述中，我们讨论了与替代肽选择相关的标准和困难，并提供了核因子 κB 通路蛋白质定量的 QconCAT 设计实例。
