de Godoy Lyris M F
Instituto Carlos Chagas, Fiocruz Parana, Rua Prof. Algacyr Munhoz Mader 3775, 81350-010, Curitiba, PR, Brazil,
Methods Mol Biol. 2014;1156:81-109. doi: 10.1007/978-1-4939-0685-7_6.
Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex mixtures, enabling characterization and comparison of cellular functional states in a proteome-wide scale. In this context, stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a simple yet powerful approach, which has been applied to address different biological questions across a variety of systems, ranging from single cells to entire multicellular organisms. In this chapter, detailed instructions for SILAC labeling yeast are provided, including a series of quality checks for evaluating labeling efficiency and procedures for determining the optimal labeling parameters for a particular yeast strain. In addition, two different complete workflows for the comprehensive mass spectrometry-based SILAC quantification of close to the entire yeast proteome are described, which can be applied to assess any biological question of interest and, therefore, can be of broad use for the researchers in the field.
基于质谱的定量蛋白质组学能够识别和定量复杂混合物中的数千种蛋白质，从而在全蛋白质组范围内对细胞功能状态进行表征和比较。在这种情况下，细胞培养中氨基酸稳定同位素标记（SILAC）已成为一种简单而强大的方法，该方法已被应用于解决从单细胞到整个多细胞生物体等各种系统中的不同生物学问题。在本章中，提供了SILAC标记酵母的详细说明，包括一系列用于评估标记效率的质量检查以及确定特定酵母菌株最佳标记参数的程序。此外，还描述了两种不同的完整工作流程，用于基于质谱对几乎整个酵母蛋白质组进行全面的SILAC定量，这些工作流程可用于评估任何感兴趣的生物学问题，因此可供该领域的研究人员广泛使用。
