Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT
Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT
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DOI：
10.1074/mcp.m600456-mcp200
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发表时间：
2007-08-01
影响因子：
7
通讯作者：
Beynon, Robert J.
中科院分区：
文献类型：
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作者：
Rivers, Jenny;Simpson, Deborah M.;Beynon, Robert J.
稳定同位素标记的蛋白质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，对于要定量的每种蛋白质，必须以相对高的成本合成稳定的同位素标记的肽。为了多重蛋白质定量，我们开发了一种方法，其中基因从头设计用于创建和表达包含蛋白质型肽串联的人工蛋白质（QconCAT）。这允许在单个实验中绝对定量多种蛋白质。本完整研究旨在定义QconCAT分析的性质、误差来源和统计行为。QconCAT蛋白被设计为含有来自鸡骨骼肌可溶性级分中存在的20种蛋白质的一种胰蛋白酶肽。将编码这些肽的优化的DNA序列连接并插入载体中用于在大肠杆菌中高水平表达。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，产生等摩尔系列的均匀标记的蛋白质型肽。在用胰蛋白酶进行蛋白水解消化之前，将通过亲和色谱法纯化并定量的标记的QconCAT蛋白以已知量加入到均质化的肌肉制备物中。如预期的，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用于多重定量的适用性。在一项完整的研究中，评估了技术差异的性质，并与生物学差异进行了比较。研究了替代电离和质谱方法，特别是LC-ESI-TOF MS和MALDI-TOF MS，用于分析蛋白质和胰蛋白酶肽。QconCAT提供了一种新的和有效的方法来精确和同时绝对定量的多个蛋白质，亚蛋白质组，甚至整个蛋白质组。
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.