Brun Virginie, Dupuis Alain, Adrait Annie, Marcellin Marlène, Thomas Damien, Court Magali, Vandenesch François, Garin Jérôme
Commissariat à l'Energie Atomique, DSV, iRTSV, Laboratoire d'Etude de la Dynamique des Protéomes, Grenoble, F-38054, France.
Mol Cell Proteomics. 2007 Dec;6(12):2139-49. doi: 10.1074/mcp.M700163-MCP200. Epub 2007 Sep 11.
Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.
诊断技术的发展和公共卫生监测需要能够对蛋白质生物标志物进行特异性识别和绝对定量的技术。除了免疫相关技术（如酶联免疫吸附测定）外，质谱（MS）因其高灵敏度和特异性而越来越受到关注。此外，只要有合适的参考标准，基于质谱的分析在定量方面极其准确。最近，使用化学合成的同位素标记标记肽对基于质谱的蛋白质进行绝对定量取得了重大进展。然而，我们在此表明，使用此类肽可能会导致严重偏差。在这项工作中，我们提出了一种创新策略（蛋白质标准绝对定量法），该策略使用体外合成的同位素标记全长蛋白质作为绝对定量的标准。由于这些蛋白质标准与目标蛋白质的生化特性完美匹配，它们可以直接添加到待分析的样品中，即使在预分级的复杂样品中也能实现对蛋白质的高度准确定量。我们的蛋白质标准绝对定量方法对生物标志物进行准确绝对定量的能力在被金黄色葡萄球菌超抗原毒素污染的水和尿液样本上得到了验证，这些毒素是具有公共卫生意义的典型生物标志物。
