Rivers Jenny, Simpson Deborah M, Robertson Duncan H L, Gaskell Simon J, Beynon Robert J
Proteomics and Functional Genomics Group, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, United Kingdom.
Mol Cell Proteomics. 2007 Aug;6(8):1416-27. doi: 10.1074/mcp.M600456-MCP200. Epub 2007 May 17.
Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.
稳定同位素标记的蛋白型肽被用作蛋白质组学中蛋白质绝对定量的替代标准。然而，对于每一种要定量的蛋白质，都必须以相对较高的成本合成一种稳定同位素标记的肽。为了实现蛋白质定量的多重分析，我们开发了一种方法，即利用从头基因设计来创建和表达包含蛋白型肽串联体的人工蛋白质（QconCATs）。这使得在单个实验中能够对多种蛋白质进行绝对定量。本完整研究旨在确定QconCAT分析的性质、误差来源和统计行为。QconCAT蛋白被设计为包含来自鸡骨骼肌可溶性部分中20种蛋白质的一个胰蛋白酶肽段。编码这些肽段的优化DNA序列被串联起来，并插入到一个载体中，以便在大肠杆菌中进行高水平表达。该蛋白质在含有用稳定同位素选择性标记的氨基酸的基本培养基中表达，从而产生一系列等摩尔的均匀标记的蛋白型肽。经亲和层析纯化并定量后的标记QconCAT蛋白，在胰蛋白酶进行蛋白水解消化之前，以已知量添加到匀浆的肌肉制剂中。正如预期的那样，QconCAT以远高于分析物蛋白质的速率被完全消化，证实了这种人工蛋白质在多重定量中的适用性。在一项完整研究中评估了技术方差的性质，并与生物学方差进行了比较。研究了用于蛋白质和胰蛋白酶肽分析的替代电离和质谱方法，特别是液相色谱 - 电喷雾 - 飞行时间质谱（LC - ESI - TOF MS）和基质辅助激光解吸电离 - 飞行时间质谱（MALDI - TOF MS）。QconCATs为精确同时绝对定量多种蛋白质、亚蛋白质组甚至整个蛋白质组提供了一种新的有效方法。
