Towfighi Parhom, Shaw Jacob, Sigdel Tara
University of California San Francisco, San Francisco, CA, USA.
Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2018;1788:157-164. doi: 10.1007/7651_2017_91.
In order to simplify a complex mixture of soluble proteins from tissues, a protocol to fractionate samples prior to two-dimensional (2D) gel electrophoresis has been developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins with DEAE-Sepharose can result in an increase in the number of detectable 2D gel spots. These gel spots are amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. The DEAE-Sepharose column fractionation acts to partition soluble proteins from cell extracts. Similarly, a SP-Sepharose column can fractionate soluble proteins and increase the number of detectable gel spots. Lastly, fractionation of cell extract with a phenyl Sepharose column can also result in an increase in the number of detectable 2D gel spots. This chapter describes an easy, inexpensive way to fractionate soluble proteins and a way to better profile proteomes.
为了简化从组织中提取的可溶性蛋白质的复杂混合物，已开发出一种在二维（2D）凝胶电泳之前对样品进行分级分离的方案。这些方法包括使用DEAE-琼脂糖、SP-琼脂糖和苯基琼脂糖色谱柱，并分别根据蛋白质的不同阴离子、阳离子和疏水特性对蛋白质混合物进行分级分离。用DEAE-琼脂糖对可溶性蛋白质进行分级分离可增加可检测到的2D凝胶斑点数量。这些凝胶斑点可通过凝胶内胰蛋白酶消化、基质辅助激光解吸/电离飞行时间质谱和肽质量指纹图谱进行蛋白质鉴定。DEAE-琼脂糖柱分级分离的作用是从细胞提取物中分离可溶性蛋白质。同样，SP-琼脂糖柱可以对可溶性蛋白质进行分级分离并增加可检测到的凝胶斑点数量。最后，用苯基琼脂糖柱对细胞提取物进行分级分离也可增加可检测到的2D凝胶斑点数量。本章介绍了一种简单、廉价的可溶性蛋白质分级分离方法以及一种更好地分析蛋白质组的方法。
