Department of Medicine, Upstate Medical University, Syracuse, NY, USA.
Blood Cells Mol Dis. 2011 Aug 15;47(2):85-94. doi: 10.1016/j.bcmd.2011.03.008. Epub 2011 May 18.
Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.
溶血性贫血是最常见的遗传性疾病之一。为了鉴定参与溶血性贫血病理生理学的候选蛋白，我们利用无标记比较蛋白质组学方法来检测正常和β-辅肌动蛋白（Add2）敲除小鼠的 RBC 差异。我们在β-辅肌动蛋白敲除 RBC 影中检测到 7 种减少的蛋白和 48 种增加的蛋白。由于溶血性贫血的特征是网织红细胞增多，我们比较了苯肼处理的小鼠的网织红细胞富集样本和未处理小鼠的成熟 RBC。无标记分析在网织红细胞富集样本中鉴定出 47 种增加的蛋白和 21 种减少的蛋白。在 Add2 敲除 RBC 中增加的蛋白中，只有 11 种也在网织红细胞中增加。在 Add2 敲除 RBC 中减少的蛋白中，β-和α-辅肌动蛋白的强度差异最大，其次是钠-氢交换体 Slc9a1。我们通过免疫印迹验证了这些质谱结果。这是溶血性贫血中 NHE-1 缺乏的第一个例子，并为导致脆弱 RBC 的机制提供了新的见解。我们使用无标记比较蛋白质组学来进行这一发现，证明了与代谢或化学同位素标记小鼠相比，这种方法的有用性。
