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    Comprehensive Proteomic Analysis of Human Erythropoiesis

    Team Patrick Mayeux

    The team of Patrick Mayeux and the proteomic platform of the Paris  Descartes University (3P5) have recently published in Cell Reports the first description of the modifications of the human erythroid cells during their differentiation

    Healthy humans produce around two million red cells each second of their lives. As other blood cells, erythrocytes originate from the multipotent hematopoietic stem cells that generate lineage-committed progenitors. Erythroid progenitors (BFU-E then CFU-E) proliferate strongly with few morphological modifications and give rise to the erythroid precursors that realize the terminal differentiation program during a limited number of cell divisions. Different types of erythroid precursors are successively produced that can be morphologically recognized: the pro-erythroblasts and basophilic, polychromatophilic and lastly orthochromatic erythroblasts. Finally, orthochromatic erythroblasts expel their nucleus to give rise to a reticulocyte that finish its maturation first in the bone marrow and then in the blood stream to produce an erythrocyte. Several teams have already reported the evolution of the transcriptome of erythroid cells during their differentiation but no general proteomic analysis of this process had been still published.

    Starting from CD34+ hematopoietic progenitors from cord blood, we modified an erythroid culture protocol to obtain homogeneous cell populations regarding their differentiation stage and we used a highly sensitive label-free proteomic method to analyze the evolution of the human erythroid cell proteome during the differentiation process. Using these methods, we quantified the absolute expression of 6,130 proteins during erythroid differentiation from BFU-E to orthochromatic erythroblasts. All the transcription factors previously known to play a specific role in erythropoiesis were quantified on a copy number per cell basis and many proteins whose expression in erythroid cells were not expected have been identified and quantified.

    In collaboration with the team of N. Mohandas (New-York Blood Center), we compared the evolution of the transcriptome and the proteome of these cells and, as usually, the found only a limited correlation. By focusing on the most divergent mRNA/protein pairs, we showed that the recently reported intron retention process that occurs at the end of the erythroid differentiation process contributed to the discrepancy between the mRNA and protein expressions.

    In the second part of the study, we determined in collaboration with the team of L. Douay (Saint-Antoine Hospital, Paris) the quantitative repartition of the proteins between the reticulocyte and the nucleus-containing particle (the pyrenocyte) after enucleation. For this study, we used an ITRAQ labeling methods that allowed to quantify the repartition of more than 1300 between each particle. Using an imaging flow cytometry method (“AMNIS”), we determined the cytoplasmic volume and the plasma membrane surface of each particle and we used these measurements to identify the proteins that have been actively sorted to one or the other particle.          

    This work is the first absolute quantification of proteins during a human complex differentiation process at the overall proteome level.  It constitutes a significant improvement in the global description of erythropoiesis and provides an important toolbox for the understanding of the erythroid differentiation process and its deregulations in pathologies leading to erythroid deficiencies like myelodysplastic syndromes, thalassemia or Diamond-Blackfan anemia.  

    Publication:

    Gautier EF, Ducamp S, Leduc M, Salnot V, Guillonneau F, Dussiot M, Hale J, Giarratana MC, Raimbault A, Douay L, Lacombe C, Mohandas N, Verdier F, Zermati Y, Mayeux P
    Comprehensive Proteomic Analysis of Human Erythropoiesis.
    Cell Rep. 2016 Aug 2;16(5):1470-84.

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