Isotope-labeled protein standards
Isotope-labeled protein standards
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DOI：
10.1074/mcp.m700163-mcp200
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发表时间：
2007-12-01
影响因子：
7
通讯作者：
Garin, Jerome
中科院分区：
文献类型：
--
作者：
Brun, Virginie;Dupuis, Alain;Garin, Jerome
诊断开发和公共卫生监测需要提供蛋白质生物标志物的特异性鉴定和绝对定量的技术。除了免疫相关技术（e。G.酶联免疫吸附测定），MS由于其高灵敏度和特异性而获得越来越多的关注。此外，只要有合适的参比标准品，基于MS的分析在定量上非常准确。最近，化学合成的同位素标记的标记肽用于基于MS的蛋白质绝对定量的使用已经导致了重大进展。然而，我们在这里表明，使用这样的肽可能会导致严重的偏见。在这项工作中，我们提出了一种创新的策略（蛋白质标准绝对定量），使用在体外合成的同位素标记的全长蛋白质作为绝对定量的标准。由于这些蛋白质标准品与目标蛋白质的生化特性完全匹配，因此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可以高度准确地定量蛋白质。我们的蛋白质标准品绝对定量方法用于生物标志物的准确绝对定量的能力被证明是被金黄色葡萄球菌超抗原毒素污染的水和尿液样品作为公共卫生利益的典型生物标志物。
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 water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.