Eisenberg-Lerner Avital, Regev Ifat, Merbl Yifat
Department of Immunology, The Weizmann Institute of Science, 234 Herzl Street, Rehovot, 7610001, Israel.
Methods Mol Biol. 2017;1647:139-152. doi: 10.1007/978-1-4939-7201-2_9.
Posttranslational modifications (PTMs) of proteins are an integral part of major cellular regulatory mechanisms dictating protein function, localization, and stability. The capacity to screen PTMs using protein microarrays has advanced our ability to identify their targets and regulatory role. This chapter discusses a unique procedure that combines functional extract-based activity assay with large-scale screening utilities of protein microarrays. This "PTM-profiling" system offers advantages in quantitatively identifying modifications in an unbiased manner in the context of specific cellular conditions. While the possibilities of studying PTMs in different settings are enormous, the immune system presents an attractive model for studying the effects of perturbations in PTMs, and specifically the ubiquitin system, as these were already implicated in both immune function and dysfunction. This chapter discusses the significance of PTM profiling in addressing basic questions in immunology. We describe detailed protocols for the preparation of functional cell extracts from immune cell cultures, following differentiation or induced signals, and screening PTMs on protein arrays, as well as basic guidelines for data analysis and interpretation.
蛋白质的翻译后修饰（PTM）是决定蛋白质功能、定位和稳定性的主要细胞调节机制的一个组成部分。利用蛋白质微阵列筛选PTM的能力提升了我们识别其靶点和调节作用的能力。本章讨论了一种独特的方法，该方法将基于功能提取物的活性测定与蛋白质微阵列的大规模筛选工具相结合。这种“PTM分析”系统在特定细胞条件下以无偏倚的方式定量鉴定修饰方面具有优势。虽然在不同环境中研究PTM的可能性巨大，但免疫系统是研究PTM扰动影响的一个有吸引力的模型，特别是泛素系统，因为它们已经与免疫功能和功能障碍有关。本章讨论了PTM分析在解决免疫学基本问题中的意义。我们描述了从免疫细胞培养物中制备功能细胞提取物的详细方案，这些提取物是在分化或诱导信号后获得的，以及在蛋白质阵列上筛选PTM的方案，还有数据分析和解释的基本指南。
