Institute for Molecular Systems Biology, Eidgenoessische Technische Hochschule, Zurich, Switzerland.
Proteomics. 2010 Oct;10(20):3657-68. doi: 10.1002/pmic.201000161.
Sickle cell disease is caused by one of the 1200 known hemoglobin variations. A single-point mutation β6(A3)Glu→Val leads to sickling of red blood cells, which in turn causes a lack of oxygen supply to tissue and organs. Although sickle cell disease is well understood, treatment options are currently underdeveloped. The only Food and Drug Administration-approved drug is hydroxyurea, an inducer of fetal γ-hemoglobin, which is known to have a higher oxygen affinity than adult hemoglobins and thus alleviates symptoms. In the search for better cures, Rhesus monkeys (Macaca mulatta) serve as models for monitoring success of induction of fetal γ-hemoglobins and with recent advances in proteomics, MS has become the leading technique to determine globin expression. Similar to humans, Rhesus monkeys possess hemoglobin variants that have not been sufficiently characterized to initiate such a study. Therefore, we developed a combined bottom-up and top-down approach to identify and characterize novel hemoglobin variants of the umbilical cord blood of Rhesus monkeys. A total of four different variants were studied: α, β, γ1 and γ2. A new α- and β-hemoglobin variant was identified, and the two previously hypothesized γ-hemoglobins were identified. In addition, glutathionylation of both γ-hemoglobin variants at their cysteines has been characterized. The combined approach outperformed either bottom-up or top-down alone and can be used for characterization of unknown hemoglobin variants and their PTMs.
镰状细胞病是由 1200 种已知的血红蛋白变异之一引起的。单点突变β6(A3)Glu→Val 导致红细胞镰状化，进而导致组织和器官缺氧。尽管镰状细胞病的发病机制已经很清楚，但目前治疗方法的开发还不够完善。唯一获得美国食品和药物管理局批准的药物是羟基脲，它是一种诱导胎儿γ-血红蛋白的物质，已知其与成人血红蛋白相比具有更高的氧亲和力，从而缓解症状。在寻找更好的治疗方法的过程中，恒河猴（Macaca mulatta）被用作监测诱导胎儿γ-血红蛋白成功的模型，并且随着蛋白质组学的最新进展，MS 已成为确定珠蛋白表达的主要技术。与人类相似，恒河猴具有尚未充分表征以启动此类研究的血红蛋白变异。因此，我们开发了一种组合的自上而下和自下而上的方法来鉴定和表征恒河猴脐带血中的新型血红蛋白变异。总共研究了四种不同的变体：α、β、γ1 和 γ2。鉴定了一种新的α-和β-血红蛋白变体，并鉴定了之前假设的两种γ-血红蛋白变体。此外，还对两个γ-血红蛋白变体的半胱氨酸进行了谷胱甘肽化修饰。组合方法的性能优于单独的自上而下或自下而上方法，可用于鉴定未知的血红蛋白变体及其 PTMs。
