Application of two-dimensional difference gel electrophoresis to studying bone marrow macrophages and their in vivo responses to ionizing radiation.

A flow cytometric protocol was developed to isolate primary bone marrow resident macrophages (CD11b((-)) Gr-1((-)) F4/80((+))) before and 24 h after 0.5 Gy gamma-irradiation from mouse strains (C57BL/6 and CBA/Ca) that exhibit significant differences in the response of their hematopoietic tissues to ionizing radiation. The proteins from these populations were analyzed using two-dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. We identified 36 macrophage proteins from 52 spots in both C57BL/6 and CBA/Ca. Thirty-three spots showed significant difference between genotypes and 16 of them corresponding to 11 proteins were identified. These included G-protein signaling 16, glucose-regulated protein 78, and lactoylglutathione lyase. We detected 16 and 18 spot changes following irradiation in C57BL/6 and CBA/Ca respectively, and in total 16 of them were identified. The identified proteins included calreticulin, lactoylglutathione lyase, regulator of G-protein signaling 16 and peroxiredoxin 5, mitochondrial precursor. The application of DIGE to primary bone marrow resident macrophages has allowed the first description of the proteome of these important components of the hematopoietic microenvironment and an analysis of their in vivo response to ionizing radiation which may shed light on the mechanism underlying the differential radiation-induced leukemogenesis exhibited within these mouse strains.