Brun Virginie, Masselon Christophe, Garin Jérôme, Dupuis Alain
CEA, DSV, iRTSV, Laboratoire d'Etude de la Dynamique des Protéomes, Grenoble, F-38054, France.
J Proteomics. 2009 Jul 21;72(5):740-9. doi: 10.1016/j.jprot.2009.03.007. Epub 2009 Mar 31.
The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical biomarker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies.
基于质谱（MS）的高通量蛋白质鉴定方法的发展，同时产生了对蛋白质定量的需求。众多基于MS的相对定量方法已致力于多个蛋白质组的广泛比较。另一方面，能够测定生物样品中蛋白质浓度的绝对定量方法，通常仅限于特定的蛋白质组。根据所选的分析程序，绝对定量方法可以提供准确而精确的估计。这些分析性能对于诸如评估临床生物标志物候选物等特定应用至关重要。根据生物分析指南，准确的分析过程需要内标和质量控制。对于基于MS的小分子分析，同位素稀释已被公认为内标标准化的参考方法。然而，依赖同位素稀释原理的蛋白质定量方法直到最近才在蛋白质组学领域得以应用。在这些方法中，向样品中加入特定量的特定蛋白水解肽的同位素标记类似物（AQUA和QconCAT策略）或蛋白质（PSAQ策略）。在本综述中，我们对这些同位素稀释方法进行了批判性概述。
