Protein phosphorylation is a central mechanism of signal transduction that both positively
and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation
states of cell signaling systems have been applied extensively in cell lines and whole
tissues to reveal critical regulatory networks, and candidate-based evaluations of
phosphorylation in rare cell populations have also been informative. However, application
of comprehensive profiling technologies to adult stem cell and progenitor populations
has been challenging, due in large part to the scarcity of such cells in adult tissues.
Here, we combine multi-color flow cytometry with highly efficient three dimensional
HPLC/mass spectrometry to enable quantitative phosphoproteomic analysis from 200,000
highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using
this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and
demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator
of its function. Our approach provides a robust platform for large-scale phosphoproteomic
analyses performed with limited numbers of rare progenitor cells. Data from our study
comprises a new resource for understanding the molecular signaling networks that underlie
hematopoietic stem cell mobilization.
