A combined top-down and bottom-up MS approach for the characterization of hemoglobin variants in Rhesus monkeys.
A combined top-down and bottom-up MS approach for the characterization of hemoglobin variants in Rhesus monkeys.
复制标题
用于表征恒河猴血红蛋白变异的自上而下和自下而上相结合的 MS 方法。
DOI：
10.1002/pmic.201000161
复制
发表时间：
2010
期刊：
影响因子：
3.4
通讯作者：
Hess,Sonja
中科院分区：
文献类型：
--
作者：
Hüttenhain,Ruth;Hess,Sonja
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→瓦尔导致红细胞镰状化，进而导致组织和器官供氧不足。虽然镰状细胞病是很好的理解，治疗方案目前还不发达。美国食品和药物管理局唯一批准的药物是羟基脲，一种胎儿γ-血红蛋白的诱导剂，已知其具有比成人血红蛋白更高的氧亲和力，从而减轻症状。在寻找更好的治疗方法时，恒河猴（Macaca mulatta）作为监测胎儿γ-血红蛋白诱导成功的模型，随着蛋白质组学的最新进展，MS已成为确定珠蛋白表达的领先技术。与人类相似，恒河猴拥有血红蛋白变体，尚未充分表征以启动此类研究。因此，我们开发了一种自下而上和自上而下相结合的方法来识别和表征恒河猴脐带血的新型血红蛋白变体。总共研究了四种不同的变体：α、β、γ1和γ2。发现了一种新的α-和β-血红蛋白变体，并发现了两种先前假设的γ-血红蛋白。此外，已表征了两种γ-血红蛋白变体在其半胱氨酸处的谷胱甘肽化。这种组合方法的性能优于单独的自下而上或自上而下，可用于表征未知血红蛋白变体及其PTM。
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.