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In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification. |
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