Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

Abstract

Reticulocytesproduced inthebonemarrowundergomaturation inthe bloodstreamto give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins weremade using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of associationwith the plasmamembrane. In addition, weused modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotinmolecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocytematuration. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.