Mass Spectrometry-compatible Subcellular Fractionation for Proteomics

We found that nuclear envelopes stabilize against surfactants in the presence of ethylene glycol (EG). We, therefore, developed a novel subcellular fractionation approach for proteomics using RIPA buffer containing EG and phase transfer surfactants. This method involves separating the cells into the cytoplasm, organelles, and nucleus, including intermediate filaments without ultracentrifugation. These fractions are directly applicable to sample preparation for shotgun proteomics as they have no mass spectrometry-incompatible chemicals, whereas those separated by traditional fractionation protocols require desalting. This protocol is successfully applied to subcellular fractionation with only 3.5 × 105 cells. Here, it was combined with phosphoproteomics and proteomics to identify phosphorylation sites regulating protein subcellular localization. In total, 59 phosphorylation sites on 42 phosphopeptides and 32 proteins showing different enrichment patterns between phosphoproteomics and the corresponding prot...