Ana Martínez-Val, Jose Manuel Rodríguez, Jesper V Olsen, Jesús Vázquez
Localization of proteins to different cell compartments is a major posttranslational regulatory mechanism that eukaryotic cells have evolved to coordinate protein homeostasis and responses to stimuli. Subcellular fractionation allows the separation of distinct protein populations from cellular compartments such as the cytosol, cytoskeleton, membrane, endoplasmic reticulum, or nucleolus. This fractionation can be followed by mass spectrometry-based proteomics to provide insights into spatiotemporal regulation or cellular protein networks. Here, we describe a high-throughput workflow based on sequential cell lysis, which enables the profiling of subcellular proteome architecture and the detection of changes in protein localization at global and posttranslational levels.