Gerber Scott A, Kettenbach Arminja N, Rush John, Gygi Steven P
Department of Genetics, Norris Cotton Cancer Center and Dartmouth Medical School, Hanover, NH, USA.
Methods Mol Biol. 2007;359:71-86. doi: 10.1007/978-1-59745-255-7_5.
The absolute quantification (AQUA) strategy provides a means to determine the precise protein or modified protein levels directly from cells or tissues. The technique is based on two major principles: stable isotope dilution theory and the use of synthetic peptides containing such stable isotopes to exactly mimic native counterparts after proteolysis. These peptides can be synthesized with modifications such as phosphorylation. methylation, and acetylation to allow for the direct, quantitative analysis of posttranslationally modified proteins. In this chapter, we discuss the development of an AQUA method and demonstrate its usefulness in the measurement of endogenous levels of the human protein separase at a functionally relevant phosphorylation site, serine 1126.
绝对定量（AQUA）策略提供了一种直接从细胞或组织中确定精确蛋白质或修饰蛋白质水平的方法。该技术基于两个主要原理：稳定同位素稀释理论以及使用含有此类稳定同位素的合成肽在蛋白水解后精确模拟天然对应物。这些肽可以进行磷酸化、甲基化和乙酰化等修饰，以便对翻译后修饰的蛋白质进行直接的定量分析。在本章中，我们讨论了一种AQUA方法的开发，并展示了其在测量人源蛋白分离酶功能相关磷酸化位点丝氨酸1126的内源性水平方面的实用性。
