Small-Scale Mass Spectrometry-Based Phosphoproteomic Analysis of Primary Hematopoietic Stem and Progenitor Cells to Identify Critical Regulators of HSPC Mobilization and Leukemogenesis

Cellular functions are largely effected by proteins, but protein-level analysis of hematopoietic stem and progenitor cell (HSPC) functions has historically been challenged by the difficulty of performing comprehensive and robust proteomic studies of rare cell populations. To confront this challenge, we developed a novel nanoscale multidimensional mass spectrometry-based phosphoproteomic platform that allows, for the first time, comprehensive and unbiased analysis of the activated protein circuits in blood stem cells, as assessed by protein phosphorylation status. We used this platform to interrogate the proteomic features responsible for the growth and maintenance of hematopoietic progenitors. Our analysis pipeline is capable of returning 12,000 unique phosphopeptide sequences (corresponding to several thousand proteins) from an input of 400,000 FACS-sorted primary mouse HSPCs. Among these phosphorylated proteins, the novel Rac-GAP Arhgap25 emerged as an important regulator of mobilization in HSPCs. Arhgap25 is phosphorylated upon treatment of HSPCs with a standard cyclophosphamide-GCSF mobilization protocol. Germline deletion of Arhgap25 in mice impairs HSPC egress from the bone marrow, both at rest and after mobilizing stimuli. These findings validate the use of this platform in the discovery of new therapeutic targets in hematopoiesis, and present a clear pathway for identifying novel targets in other rare subsets of human progenitor cells, including leukemia stem cells.