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 (MS)-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 proteomics
were identified, which are potential candidate sites to regulate the
protein subcellular localization, including serine 706 on CD44 and
serine 22 on lamin A/C.