Bioanalytical platform for quantitative interrogation of signaling pathways in HSPCs. Two × 10⁵ highly purified primary murine HSPCs were sorted by flow cytometry, lysed, and trypsin-digested, followed by Fe³⁺NTA-IMAC phosphopeptide enrichment and isotope labeling with TMT reagents. Phosphopeptides were quantified by fully automated 3-D RP-SAX-RP chromatography, coupled to a ThermoFisher Orbitrap mass spectrometer. Data analysis and visualization was performed using a combination of multiplierz and R scripts. LC-MS, liquid chromatography–MS.