Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Germany.
Electrophoresis. 2012 Aug;33(16):2537-45. doi: 10.1002/elps.201200151.
The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.
细胞质红细胞（RBC）蛋白质组的分析受到细胞内大量血红蛋白的影响，这使得低丰度细胞质蛋白的检测变得复杂。在这项研究中，提出了一种替代的血红蛋白 depleted RBC 裂解物制备方法，该方法与下游 2D PAGE 分析和 Orbitrap MS 相结合。血红蛋白的去除是通过使用 HemoVoid（TM）耗尽试剂来完成的，该试剂通过同时降低样品中的血红蛋白浓度，非常有效地富集低丰度蛋白质。在定义了包括特异性/选择性、精密度和线性在内的选定样品制备方案特征后，生成了细胞质 RBC 蛋白质组的 2D 参考图谱（pH 4-7），并鉴定了总共 189 种不同的蛋白质。因此，所提出的方法被证明非常适合制备 RBC 细胞质可重复的高分辨率 2D 蛋白质图谱，并为未来研究细胞溶质 RBC 蛋白质组的疾病或储存诱导变化提供了有用的工具。
