Ericsson Christer, Peredo Inti, Nistér Monica
Department of Oncology-Pathology, Karolinska Institute, Cancer Center Karolinska, R8:05, Karolinska University Hospital-Solna, 171 76 Stockholm, Sweden.
Acta Oncol. 2007;46(1):10-20. doi: 10.1080/02841860600847061.
Optimal standard conditions for protein extraction and solubilization from frozen tissue samples have been examined. Quantitative differences in specific protein amounts or post-translational modifications underlie many, if not all, disease states. Maximal and standardized extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis, and to make the best use of a precious resource. Minimal degradation of the protein amino acid backbone, or of phosphorylated amino acid side chains, during sample preparation is essential to preserve the analytical utility of the extract. We have investigated parameters of brain tissue disintegration, and of extraction/solubilization temperature, time and volume and have reached 98% extraction of brain tissue, corresponding to about 100 microg protein per mg tissue wet weight, by an SDS-based method: Tissue disintegration in the frozen state, by ball mill grinding followed by extraction and solubilization in 2% SDS for 10 min, at 70 degrees C, in a volume corresponding to ten times the tissue wet weight, with shaking. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. Moreover, endogenous enzymes can be inhibited by incubation at high pH. The resulting protein extracts can be used for both one-dimensional SDS gel-electrophoresis and for two-dimensional isoelectric focusing/SDS electrophoresis. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification and quantitation from cryopreserved clinical samples are desirable.
已对从冷冻组织样本中提取和溶解蛋白质的最佳标准条件进行了研究。特定蛋白质数量或翻译后修饰的定量差异是许多（即便不是所有）疾病状态的基础。从患病或健康组织中最大程度地标准化提取和溶解蛋白质，对于使整个蛋白质组可用于蛋白质组学分析以及充分利用珍贵资源非常重要。在样品制备过程中，蛋白质氨基酸主链或磷酸化氨基酸侧链的降解最小化对于保持提取物的分析效用至关重要。我们研究了脑组织破碎以及提取/溶解温度、时间和体积等参数，并通过基于SDS的方法实现了98%的脑组织提取率，相当于每毫克组织湿重约100微克蛋白质：在冷冻状态下通过球磨研磨进行组织破碎，然后在70℃下于相当于组织湿重10倍的体积中用2% SDS提取和溶解10分钟，并进行振荡。用SDS样品缓冲液处理可抑制蛋白酶和磷酸酶活性。此外，通过在高pH下孵育可抑制内源性酶。所得蛋白质提取物可用于一维SDS凝胶电泳和二维等电聚焦/SDS电泳。所提出的标准方案在需要从冷冻保存的临床样本中进行蛋白质提取、溶解、鉴定和定量的情况下具有广泛应用的潜力。
