Signore Michele, Manganelli Valeria
RPPA Unit, Proteomics Area, Core Facilities, Istituto Superiore di Sanità, Rome, Italy.
Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy.
Methods Cell Biol. 2022;171:33-61. doi: 10.1016/bs.mcb.2022.04.004. Epub 2022 Jul 22.
The scenario of proteogenomics is rapidly evolving and novel technologies are enabling comprehensive molecular exploration down to single cells. Likewise, digital (immuno-)assays are revolutionizing the field of biomarker detection and have reached the grade for population-level screenings with single-molecule sensitivity. Nonetheless, cost- and time-effective, high-throughput targeted phospho-proteomics at a preclinical stage still relies on ad hoc microarray platforms, such as the Reverse-Phase Protein microArrays (RPPA). Although this technique requires specific knowledge and equipment and different laboratories worldwide have implemented alternative methodological strategies, the application of RPPA to biomarker discovery has proven successful on diverse types of samples, including tissues and biological fluids as well as nanovesicles and in vitro cultured lines. Among these, cancer stem(-like) cells (CSC) represent an ideal experimental model system for preclinical discovery and definition of novel drug targets. The present methodological article provides the basic knowledge and steps on how to deploy an RPPA analysis with specific reference to an ideal experimental setup of drug testing on CSC.
蛋白质基因组学的研究现状正在迅速发展，新技术使得对单细胞进行全面的分子探索成为可能。同样，数字（免疫）检测正在彻底改变生物标志物检测领域，并已达到可进行具有单分子灵敏度的人群水平筛查的程度。尽管如此，临床前阶段具有成本效益且高通量的靶向磷酸化蛋白质组学仍然依赖于特定的微阵列平台，例如反相蛋白质微阵列（RPPA）。虽然该技术需要特定的知识和设备，并且世界各地不同的实验室已经采用了替代的方法策略，但RPPA在生物标志物发现中的应用已在包括组织、生物体液、纳米囊泡和体外培养细胞系在内的多种类型样本上证明是成功的。其中，癌症干细胞（样）细胞（CSC）是临床前发现和定义新型药物靶点的理想实验模型系统。本文提供了关于如何进行RPPA分析的基础知识和步骤，并特别参考了在CSC上进行药物测试的理想实验设置。
