短胶法预处理含高浓度去垢剂的蛋白样品

Short gel method for pretreatment of protein samples with high concentration of detergent

摘要:

蛋白质组学研究中常使用含高浓度去垢剂的缓冲液以提高蛋白提取效率，但这种缓冲液会对下游蛋白质定量、酶解、质谱分析等步骤造成干扰。短胶法可用于含高浓度去垢剂样品的预处理，文中进一步评价了短胶法 (Short gel) 用于含高去垢剂的蛋白样品定量和预处理的效果。使用牛血清白蛋白作为标准蛋白分析了短胶法定量的线性范围，结果发现短胶法定量的线性范围为1?8 μg，提示可对这一范围内的样品蛋白进行定量检测。对标准蛋白定量的线性度达到0.999，且重复性好，不容易受到蛋白表达模式变化的影响，结果可靠。短胶法定量的蛋白可通过胶内酶解，直接进行液相色谱-质谱联用 (Liquid chromatography-tandem mass spectrometry, LC-MS/MS) 分析，质谱信号较好。结果提示直接用短胶法预处理含高浓度去垢剂的蛋白样品，值得在蛋白质组学研究中推广。

Abstract:

In proteomic research, to improve protein solubility of membrane proteins and nuclear proteins, buffers containing high concentration of detergent, such as 4% SDS, were widely used. However, high concentration of detergent might severely interfere with the downstream proteomic analysis, including protein quantitation and trypsin digestion. To improve the proteomic compatibility of buffers with high concentration of detergent, we used short gel method to pretreat buffers containing detergent. Protein samples were first separated by a short (2?2.5 mm) SDS-PAGE electrophoresis, and proteins were quantitated by comparing with bovine serum albumin standards via optical density analysis. The gel was then cut and peptides were recovered using in-gel digestion. The quantitative linearity range of this method was 1 to 8 μg. The quantitation was accurate and reproducible. After short gel analysis, recovered peptides generated high mass spectrometry signals. In conclusion, short gel method eliminated the interference of high concentration detergent in the proteomics analysis, and it was suitable for protein samples’ pretreatment, and was worth to apply in proteomic research.