大鼠骨组织蛋白质组样品提取方法的建立

The Method of Extracting the Protein Specimen from Rat Bone for Further Proteomics Analysis

蛋白质组学是目前研究的重要热点.该文拟建立大鼠骨组织蛋白质样品提取的最佳方法,用于双向电泳分离,为进一步进行蛋白组学分析奠定基础.实验对3种不同方法制备的样本进行同一条件的二维电泳(2- DE)图谱,比较2- DE的结果.结果发现,丙酮沉淀法最佳.这些结果提示,只有结合骨组织成分特征与双向电泳的具体要求,选择恰当的样品提取液,采用合理的提取步骤,获得蛋白质样品,才能得到清晰高分辨率的电泳图谱,满足蛋白质组学分析需要.

Proteomics is a challenging field that has been growing rapidly in the postgenomic era.Protein preparation is a key procedure for two-dimensional electrophoresis (2-DE) proteomics.In the current study,three procedures of protein sample preparation,namely basic,dialysis and acetone methods,were compared for establishment of an optimal method to extract proteins from rat bone,based on 2-DE maps from protein samples prepared by the three methods.Of the three methods,acetone method is the best one.Using this method to purify proteins from rat bone,high quality of peptide mass fingerprint can be achieved.The procedure of the method is as follows.Prepared bone (1.0 g) was placed in an earthen bowl to grind into powder in liquid nitrogen and then 3 mL ice-cold buffer (1.44 g urea,0.46 g sulfocarbamide,0.12 g CHAPS,0.047 g DTT,0.015 g Tris-base,30 μL 100 mmol/L PMSF) was added to it.After stored at 4℃ overnight,these samples were centrifuged at 12 000 g for 30 min.The supernatant was dialyzed against 0.01 mol/L PBS for 24 h.Then ten times of volumes of cooled acetone were added to the specimen for the concentration of proteins.Followed by the store at -20℃ for 2 h,the proteins were obtained by centrifugation and then dry at 4℃.Before the isoelecric focusing,the sample was redissolved in the buffer (7 mol/L urea,4% CHAPS,100 mmol/L DTT,40 mmol/L Tris,2 mol/L sulfocarbamide,0.5% ampholyte (pH 5~7),1 mmol/L PMSF) at the room temperature for 2 h.The results may provide a valuable method of sample preparation for study of bone proteomics.