Maaß Sandra
Department of Microbial Proteomics, Institute for Microbiology, University Greifswald, Greifswald, Germany.
Methods Mol Biol. 2018;1841:141-162. doi: 10.1007/978-1-4939-8695-8_11.
Absolute protein quantification for the analysis of proteome dynamics is more and more required by the scientific community. Therefore a number of methods have recently been reported that aim at determining concentrations of single proteins in complex samples, all of them having their advantages and limitations. However, for all of these methods an accurate and protein unspecific determination of the total protein amount in a given sample is urgently needed. Here a ninhydrin-based assay established to reach this goal is described. Moreover, an optimized protocol for protein digestion is an inevitable prerequisite for all mass spectrometry-based approaches aiming at absolute protein quantification. In this chapter, various aspects are described which have to be considered during validation of a suitable digestion method and a detailed protocol is presented that can be applied to the digestion of soluble proteins originated from microbes.In order to provide an absolute protein quantification workflow applicable for small scale and large scale approaches, a step-by-step guide is provided for the so-called AQUA-strategy (AQUA = absolute quantification), including selection of suited standard peptides, the development of optimized MS methods and the determination of absolute protein concentration using stable isotope dilution and selected reaction monitoring (SID-SRM). Subsequently, a workflow is introduced that combines targeted mass spectrometry and two-dimensional polyacrylamide gel electrophoresis for the large-scale determination of absolute protein amounts.
科学界对用于蛋白质组动力学分析的绝对蛋白质定量的需求日益增加。因此，最近报道了许多旨在测定复杂样品中单一蛋白质浓度的方法，所有这些方法都有其优点和局限性。然而，对于所有这些方法而言，迫切需要对给定样品中的总蛋白量进行准确且不依赖蛋白质特异性的测定。本文描述了一种基于茚三酮的测定方法，该方法旨在实现这一目标。此外，优化的蛋白质消化方案是所有基于质谱的绝对蛋白质定量方法不可避免的前提条件。在本章中，描述了在验证合适的消化方法时需要考虑的各个方面，并给出了一个详细的方案，该方案可应用于源自微生物的可溶性蛋白质的消化。为了提供适用于小规模和大规模方法的绝对蛋白质定量工作流程，针对所谓的AQUA策略（AQUA =绝对定量）提供了分步指南，包括选择合适的标准肽、开发优化的质谱方法以及使用稳定同位素稀释和选择反应监测（SID-SRM）测定绝对蛋白质浓度。随后，引入了一种工作流程，该流程结合了靶向质谱和二维聚丙烯酰胺凝胶电泳用于大规模测定绝对蛋白量。
