Schmidt Carla, Urlaub Henning
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Methods Mol Biol. 2012;893:249-65. doi: 10.1007/978-1-61779-885-6_17.
Mass spectrometry (MS) is a powerful tool for protein identification and has therefore become indispensable for proteome studies. In recent years, simple protein identification by MS has become routine, and more attention has been devoted to the MS-based investigation of posttranslational modifications and the quantification of proteins and peptides. Numerous methods and techniques for relative quantification of proteins by MS have emerged and have been applied successfully to answer various questions of protein abundance. Absolute quantification is often applied in clinical research and biomarker discovery, but has also been used to determine protein stoichiometries in protein complexes. However, the number of methods available for absolute quantification is still restricted and often requires the generation of standard peptides containing amino acids labeled with stable isotopes, although label-free approaches are also gaining importance. Complete hydrolysis of the proteins to be quantified is known to be one of the prerequisites for reliable absolute quantification, and selection and suitability of the standard peptides are critical factors in the planning of a quantitative study. Along the different methods to read out quantitative signals by MS, multiple reaction monitoring (MRM) has proven to be most suitable, with a wide linear range. However, analysis by MRM is a targeted approach and each case requires the individual design of suitable assays, which is a time-consuming step during the preliminary analysis. In this chapter, we present various protocols for in-solution hydrolysis, manual selection of suitable standard peptides, and design of MRM transitions.
质谱（MS）是用于蛋白质鉴定的强大工具，因此已成为蛋白质组研究中不可或缺的手段。近年来，通过质谱进行简单的蛋白质鉴定已成为常规操作，人们更多地关注基于质谱对翻译后修饰的研究以及蛋白质和肽段的定量分析。众多通过质谱对蛋白质进行相对定量的方法和技术不断涌现，并已成功应用于解答各种蛋白质丰度问题。绝对定量常用于临床研究和生物标志物发现，但也被用于确定蛋白质复合物中的蛋白质化学计量。然而，可用于绝对定量的方法数量仍然有限，通常需要生成含有用稳定同位素标记氨基酸的标准肽段，尽管无标记方法也日益重要。已知对要定量的蛋白质进行完全水解是可靠的绝对定量的前提条件之一，标准肽段的选择和适用性是定量研究规划中的关键因素。在通过质谱读取定量信号的不同方法中，多反应监测（MRM）已被证明是最合适的，具有较宽的线性范围。然而，MRM分析是一种靶向方法，每个案例都需要单独设计合适的检测方法，这在初步分析过程中是一个耗时的步骤。在本章中，我们介绍了多种用于溶液内水解、手动选择合适的标准肽段以及设计MRM跃迁的方案。
