The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications
The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications
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DOI：
10.1016/j.ymeth.2004.08.018
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发表时间：
2005-03-01
期刊：
影响因子：
4.8
通讯作者：
Gygi, SP
中科院分区：
文献类型：
--
作者：
Kirkpatrick, DS;Gerber, SA;Gygi, SP
生物质谱法的进步导致了许多用于大规模定量细胞内蛋白质表达水平的策略的发展。蛋白质表达的这些测量最通常通过将稳定同位素差异掺入细胞蛋白质来完成。已经证明了稳定同位素定量方法的几种变化，不同的同位素组成和掺入策略。通常，这些方法中的大多数仅建立表达蛋白的相对定量。为了解决这一问题，开发了绝对定量（AQUA）策略，用于精确测定蛋白质表达和翻译后修饰水平。AQUA方法依赖于使用在消化期间以已知浓度引入细胞裂解物的合成内标肽。这种AQUA肽精确地模拟了靶蛋白的蛋白水解过程中产生的肽，除了它富含某些稳定同位素。在串联质谱仪中通过选择反应监测（SRM）实验分析蛋白水解的样品导致天然肽和同位素标记的AQUA内标肽的直接检测和定量。作为一个例子，开发和应用的方法来测量胰蛋白酶肽代表的量的多聚泛素链形成通过赖氨酸48（K-48）。该方法的简单性和灵敏度，加上串联质谱仪的广泛可用性，使AQUA策略成为直接从细胞裂解物中测量蛋白质和翻译后修饰水平的非常有用的程序。(c)2004年爱思唯尔公司All rights reserved.
Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K-48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates. (c) 2004 Elsevier Inc. All rights reserved.