Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes
Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes
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DOI：
10.1182/bloodadvances.2018023515
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发表时间：
2018-10-23
期刊：
影响因子：
7.5
通讯作者：
Mayeux, Patrick
中科院分区：
文献类型：
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作者：
Gautier, Emilie-Fleur;Leduc, Marjorie;Mayeux, Patrick
骨髓中产生的网织红细胞在血液中成熟，产生红细胞。尽管循环红细胞的蛋白质组已经成为几个报告的主题，但用于这些研究的细胞群体从未完全缺乏网织红细胞。在我们当前的研究中，我们使用高度纯化的红细胞和网织红细胞群体来量化每个细胞群体中蛋白质的绝对表达水平。红细胞和网织红细胞在多步骤过程中纯化，包括纤维素层析、Percoll梯度离心和噻唑橙子标记后的荧光细胞分选。通过质谱法分析来自全细胞和红细胞质膜（血影）的蛋白质，从而鉴定和定量2077种蛋白质，包括654种网织红细胞特异性蛋白质。使用细胞的平均红细胞血红蛋白含量作为标准，对这些蛋白质进行绝对定量。对于每种蛋白质，我们计算了网织红细胞成熟末期的损失百分比和与质膜结合的百分比。此外，我们使用了修饰的腺苷三磷酸和腺苷二磷酸分子，使生物素分子转移到激酶的催化位点，以分离红细胞中的活性激酶，并确定了75种蛋白激酶的绝对表达及其在网织红细胞成熟过程中表达的修饰。我们的研究结果代表了第一个绝对定量的蛋白质，在正常红细胞中特异性表达，没有检测到网织红细胞的污染。因此，我们的研究结果代表了未来的病理性红细胞蛋白质组学分析的参考数据库。
Reticulocytesproduced in the bone marrow undergo maturation in the bloodstream to 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 were made 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, we used 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.