Rinalducci Sara, Longo Valentina, Ceci Luigi R, Zolla Lello
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
J Mass Spectrom. 2015 Feb;50(2):326-35. doi: 10.1002/jms.3531.
One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.
血库储存的红细胞（RBCs）的一个特征是从盘状不可逆地转变为类似球形红细胞的形态，伴有膜扰动和细胞骨架紊乱。因此，鉴定细胞骨架与脂质双层之间蛋白质 - 蛋白质相互作用中与储存相关的修饰，可能有助于阐明储存红细胞机械性能改变所涉及的分子机制。在此，我们报告了通过基于无标记质谱（MS）的实验，分析在标准血库条件下储存0、21和35天后的红细胞所获得的结果。我们能够定量测量属于β - 血影蛋白、锚蛋白 - 1、α - 内收蛋白、血影蛋白、血型糖蛋白A和血型糖蛋白C蛋白的关键磷酸肽的磷酸化水平变化。数据已通过蛋白质印迹法和伪多反应监测（MRM）进行了验证。尽管每个磷酸肽都显示出独特的趋势，但在储存期间观察到磷酸化水平急剧增加。磷酸肽图谱和结构建模分析表明，磷酸化残基位于维持膜结构完整性的蛋白质功能域中。连同先前通过电子显微镜获得的形态学证据，我们的结果似乎表明，21天的储存可能是导致血液储存期间观察到的红细胞变形性降低的分子过程的关键点。因此，这些发现可能有助于理解和预防负责保存红细胞输血后存活的形态学相关机制。
