Deep Proteomic Analysis of Human Erythropoiesis

*the first two authors are co-first authors

Introduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process.

Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1,  transcription factors, heme biosynthesis enzymes followed the expected pattern.

To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins.

Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis.