蛋白组学研究中胰腺组织匀浆化方法的建立

Establishment of homogenization method of pancreatic tissue in the study of proteomics

目的:通过不同匀浆化方法提取大鼠胰腺组织总蛋白,建立胰腺组织匀浆化的标准方法。方法:用液氮研磨分别配合反复冻融法、超声破碎法进行高速分散器分别配合反复冻融法、超声破碎法进行匀浆化胰腺组织,总蛋白提取试剂盒提取胰腺组织总蛋白,测量蛋白浓度并用体积排阻色谱(SEC)分离后分别进行基质辅助激光电离飞行时间质谱(MALDI-TOF-MS)做蛋白全谱。结果:液氮研磨分别配合反复冻融法、超声破碎法,高速分散器分别配合反复冻融法、超声破碎法总蛋白提取量分别为每0.1g胰腺组织:(6.7±0.5)mg、(6.9±0.2)mg、(5.4±0.2)mg、(5.6±0.2)mg,MALDI-TOF-MS蛋白全谱的谱峰数分别为304.2±22.3、331.6±30.5、263.8±19.7、289.8±12.2个。结论:液氮研磨配合超声破碎法与其他3种方法相比具有匀浆化彻底、蛋白不易变性等优点。

Objective：Contrast different homogenization method to extract total protein of pancreatic tissue to find the best method to homogenize pancreatic tissue. Methods：The pancreatic tissue of health SD rat were homogenized by different method and extract the total protein of pancreatic tissue by using total protein extract kit then the protein level was measured and the protein was abruptio using SEC - RPLC, detected MALDI - TOF - MS respectively. Results ：The extraction amount of total protein with liquid nitrogen grind combined with freeze - thaw homogenization and liquid nitrogen grind combined with ultrasonic homogenization methods was （6.7 ± 0.5 ） mg, （6.9 ±0.2） mg and the number of MALDI - TOF MS spectra peak was （304.2 ± 22.3 ） and （331.6 ±30.5 ） respectively. The extraction amount of total protein with mechanical homogenization combined with freeze - thaw homogenization and mechanical homogenization combined with ultrasonic homogenization methods was （5.4 ±0.2）mg, （5.6 ±0. 2）mg and the number of MALDI -TOF MS spectra peak was （263.8 ± 19.7） and （289.8 ± 12.2） respectively. Conclusion：The liquid nitrogen grind combination ultrasonic homogenization method is the best homogenize method of pancreatic tissue and possess tissue chemical property compared with others.