Mitochondrial Proteome from Human Peripheral Blood Cells.

Abstract

Mitochondria play a special role in iron metabolism as the site of heme synthesis for hemoglobin. Mitochondria also function in cellular respiration, apoptosis, amino acid synthesis, Fe-S cluster formation and repair, and redox homeostasis; different blood cell lineages depend on some or all of these diverse mitochondrial functions. Mitochondrial abnormalities in hematopoietic stem cells might manifest themselves in proteomes of all the hematopoietic lineages. Therefore, we have begun characterization of mitochondria from different peripheral blood cell populations: platelets, lymphocytes, neutrophils and reticulocytes with the objective of comparing their function and proteomes in normals and in certain disease states. The procedures utilized as starting material a blood draw of approximately 80 ml from normal volunteers. The peripheral blood samples were separated by centrifugation and Hypaque density gradient into platelet, mononuclear cell, neutrophil and red cell populations. The red cells were further sorted by density gradient and magnetic cell sorting with specific CD71 microbeads to obtain enrichment of reticulocytes (reticulocytes retain their mitochondria and lose these upon maturation into mature red cells). The various cell fractions were evaluated by cell counting, flow cytometry and staining for morphology and identification. In accordance with differences in size and surface characteristics of these cell types, different procedures for cell rupture were utilized: shearing with a home-made device using ball bearings (mononuclear cells, neutrophils), nitrogen cavitation (platelets) and hypotonic shock (reticulocytes). Mitochondria were prepared by differential centrifugation and Percoll density gradient separation. The mitochondria were evaluated by fluorescence microscopy, flow cytometry, marker enzyme activity (succinate dehydrogenase) and Western blotting with compartment-specific antibodies. Mitochondrial protein profiles were obtained using 2-dimensional gel electrophoresis coupled to mass spectrometry. From 80 ml blood, 50 million lymphoctes were obtained equivalent to 150 microgram mitochondrial protein and 10 fold enrichment of succinate dehydrogenase activity. In parallel, K562 cell mitochondria were studied. The imaging analysis revealed significant differences in the protein patterns due to hematopoietic cell lineage. This work seeks to establish a proteomic database of shared and distinct erythroid, myeloid and lymphoid mitochondrial proteins that will form the basis of future studies of blood diseases in which perturbations of mitochondrial proteins are expected to occur. We are especially interested in examining the mitochondrial proteome and correlating with mitochondrial function in myelodysplasia and sideroblastic anemia.