Centre for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK.
STAR Protoc. 2021 Apr 10;2(2):100462. doi: 10.1016/j.xpro.2021.100462. eCollection 2021 Jun 18.
Actin-rich protrusions are membrane extensions generated by actin polymerization that drive mesenchymal-like cell migration. Characterization of protrusions proteome is crucial for understanding their function. We present a complete step-by-step protocol based on microporous filter-based fractionation of protrusive cellular domains coupled with sample preparation for quantitative proteomics, mass spectrometric data acquisition, and data analysis. This protocol enables purification, quantification, and analysis of the distribution of proteins present in protrusions and cell bodies. For complete details on the use and execution of this protocol, please refer to Dermit et al. (2020).
富含肌动蛋白的突起是由肌动蛋白聚合产生的细胞膜延伸，驱动间充质样细胞迁移。突起蛋白组的特征对于理解其功能至关重要。我们提出了一个完整的基于微孔过滤的突起细胞区域分级分离的分步协议，该方法与定量蛋白质组学、质谱数据采集和数据分析的样品制备相结合。该方案能够纯化、定量和分析存在于突起和细胞体中的蛋白质的分布。有关此方案的使用和执行的完整详细信息，请参阅 Dermit 等人。（2020 年）。
