Absolute quantitation of protein posttranslational modification isoform.
Absolute quantitation of protein posttranslational modification isoform.
复制标题
蛋白质翻译后修饰亚型的绝对定量。
DOI：
10.1007/978-1-4939-2648-0_8
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发表时间：
2015
影响因子：
--
通讯作者：
Li Ning
中科院分区：
文献类型：
--
作者：
Yang Zhu;Li Ning
质谱技术已广泛应用于复杂生物样品中蛋白质的定性和定量分析。由于在构建各种生物表型和现象的分子系统的数学模型中需要蛋白质的绝对量的数量，因此已经采用了许多定量蛋白质组学方法来使用质谱法测量蛋白质的绝对量。液相色谱-串联质谱法（LC-MS/MS）与内标肽，稳定同位素编码肽系列稀释法起源于分析化学领域，目前已成为绝对定量蛋白质组学研究中广泛应用的方法。这种方法提供了越来越多的高置信度的绝对蛋白质定量结果。由于翻译后修饰（PTM）调节蛋白质的生物学活性的定量研究是生物科学的关键，每个异构体可能会贡献一个独特的生物学功能，降解，和/或亚细胞定位，蛋白质PTM异构体的绝对定量已变得更加相关的生物学意义。为了准确地获得蛋白质的PTM同种型的绝对细胞量，应考虑蛋白质分级分离、蛋白质富集和蛋白水解消化产率的影响，并且必须在将差异稳定的同位素编码的PTM肽标准品掺入样品肽中之前校正这些影响。在稳定同位素标记的肽标准品的辅助下，绝对定量后修饰蛋白质亚型（AQUIP）方法考虑了所有这些因素，并根据目标蛋白质的绝对量和蛋白质位点的PTM占有率确定蛋白质PTM亚型的绝对量。通过定量总细胞蛋白中少数PTM位点非依赖性肽的绝对量及其肽产率来推断目标蛋白的绝对量。通过测量转基因生物体中表达的或使用亲和纯化直接从生物体分离的高度纯化的蛋白质样品中PTM和非PTM肽的绝对量来实现PTM占有率测定。最终从这两个变量计算总细胞蛋白提取物中每种PTM同种型的绝对量。按照这种方法，质谱仪给出的离子强度用于计算肽的量，然后从中推导出蛋白质同种型的量。在这一章中，我们描述了实验设计的基本原则和AQUIP方法中使用的程序。该定量方法基本上采用稳定的同位素标记的肽标准品和从标记的目的重组蛋白的亲和纯化。其他定量策略和纯化技术与此方法进行了讨论。
Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM-site-independent peptides in the total cellular protein and their peptide yields. The PTM occupancy determination is achieved by measuring the absolute amounts of both PTM and non-PTM peptides from the highly purified protein sample expressed in transgenic organisms or directly isolated from an organism using affinity purification. The absolute amount of each PTM isoform in the total cellular protein extract is finally calculated from these two variables. Following this approach, the ion intensities given by mass spectrometers are used to calculated the peptide amounts, from which the amounts of protein isoforms are then deduced. In this chapter, we describe the principles underlying the experimental design and procedures used in AQUIP method. This quantitation method basically employs stable isotope-labeled peptide standards and affinity purification from a tagged recombinant protein of interest. Other quantitation strategies and purification techniques related to this method are also discussed.
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