Üresin, N.; Petrosius, V.; Fernandez, P. A.; Furtwängler, B.; Schoof, E. M.; Porse, B. T.

Proteins play a key role in defining cellular phenotypes, yet comprehensive proteomic analysis often requires substantial input material, posing challenges in studying rare populations in complex cell systems. Here, we present an accessible, label-free low-input proteomics workflow that allows for comprehensive proteome coverage reminiscent of classical bulk samples from only 500 cells and showcase its application in murine hematopoiesis. With this approach, we construct a proteomic map of hematopoietic stem and progenitor cell (HSPC) populations isolated by fluorescence-activated cell sorting (FACS) from the bone marrow of a single mouse, identifying approximately 7,000 proteins per cell population. Our study recapitulates the differentiation trajectories along the megakaryocytic-erythroid and granulocytic-monocytic lineages. We specifically focus on the dynamics of transcriptional regulators and provide insights into both known and novel population-specific factors. Furthermore, we extend our exploration to the most primitive stem and progenitor compartment, and identify ADP-Ribosyltransferase ART4 (CD297) as a novel cell surface marker that can potentially be used to enrich for long-term hematopoietic stem cells (LT-HSC). The low-input proteomics workflow presented here holds promise for overcoming the challenges associated with analyzing proteomes of rare cell populations, thereby paving the way for broader applications in biomedical research.