In a publication in eLife, Ly et al. report a workflow combining FACS and MS-based proteomics to analyse protein abundance and phosphorylation changes proteome-wide across the mitotic cell division cycle, including resolution of mitotic subphases. The approach, called PRIMMUS or proteomics of intracellular immunolabelled cell subsets, combines intracellular immunostaining, FACS, and mass spectrometry to isolate and analyse cell subsets defined by intracellular markers of cell cycle progression. Cytometric separation avoids potential artefacts associated with synchronization induced by metabolic arrest, specific and non-specific effects of small molecule kinase inhibitors, and/or spindle poisons. Key findings include the identification of a set of phosphorylations called ‘early risers’ that increase during G2 phase, and ~100 proteins showing decreased abundances during early mitosis, including RRM2. These findings were further corroborated with cell biology experiments performed in collaboration with Pat Wadsworth at University of Massachusetts, Amherst, and Emma Lundberg at the Human Protein Atlas.

Read eLife publication here.