Proteomic analysis of RBC membrane protein degradation during blood storage
Proteomic analysis of RBC membrane protein degradation during blood storage
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DOI：
10.1021/pr070179d
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发表时间：
2007-08-01
影响因子：
4.4
通讯作者：
Zolla, Lello
中科院分区：
文献类型：
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作者：
D'Amici, Gian Maria;Rinalducci, Sara;Zolla, Lello
使用二维凝胶电泳和质谱分析来识别在存在或不存在蛋白酶抑制剂的情况下，在大气氧下储存一段时间后红细胞膜中蛋白质谱的变化。在前 7 天的储存过程中，观察到分子量较低（范围在 7 至 15 kDa 之间）的新斑点，而随着时间的推移，出现更多碎片和高分子质量聚集体，表现为凝胶上部的涂抹。一些蛋白质的变化被证明是等电点的变化，这是化学氧化的结果。所有这些新斑点都是由于活性氧（ROS）攻击蛋白质而产生的。蛋白质鉴定表明大多数修饰的蛋白质位于细胞骨架中。在储存的前 7 天期间，在条带 4.2 中普遍观察到氧化降解，在条带 4.1 和 3 以及血影蛋白中观察到较小程度的氧化降解。 14 天后，出现了来自 β-肌动蛋白、3-磷酸甘油醛脱氢酶、条带 4.9 和锚蛋白等的新片段。还检测到了初步的蛋白质-蛋白质交联产物，包括α和β血影蛋白。交联产物随着时间的推移而增加。当除去氧气并将血液储存在氦气下时，蛋白质降解大大减少。有趣的是，很少有斑点与酶活性相关，并且当存在氧气时它们的数量更多，这表明某些蛋白酶可能是氧依赖性的。
Two-dimensional gel electrophoresis and mass spectrometry were used to identify protein profile changes in red blood cell membranes stored over time under atmospheric oxygen, in the presence or absence of protease inhibitors. New spots with lower molecular masses, ranging between 7 and 15 kDa were observed during the first 7 days storage, while over time, further fragments and highmolecular-mass aggregates appeared, seen as a smearing in the upper part of the gel. Some of the protein changes turned out to be shifts in isoelectric point, as a consequence of chemical oxidations. All these new spots were generated as a result of protein attack by reactive oxygen species (ROS). Protein identification revealed that most of the modified proteins are located in the cytoskeleton. During the first 7 days of storage, oxidative degradation was observed prevalently in band 4.2, to a minor extent in bands 4.1 and 3, and in spectrin. After 14 days, there were new fragments from beta-actin, glyceralclehyde-3-phosphate dehydrogenase, band 4.9, and ankyrin, among others. Preliminary protein-protein cross-linked products, involving alpha and beta spectrin, were also detected. The cross-linked products increased over time. Protein degradation was greatly reduced when oxygen was removed and blood was stored under helium. Interestingly, very few spots were related to enzyme activity, and they were more numerous when oxygen was present, suggesting that some proteases may be oxygen-dependent.