一种改进的凝胶分离后蛋白质溶液酶解方法

An Improved Method for In-solution Digestion of Gel Separated Proteins

报道了改进的凝胶电泳分离后蛋白质溶液酶解方法。将凝胶分离后的蛋白转移到PVDF膜上,直接用碳酸氢铵中和从膜上的蛋白洗脱溶液至弱碱性,并在此溶液中直接进行蛋白酶解。方法避免了胶内杂质对质谱鉴定蛋白质的干扰。用标准蛋白进行了验证,并在此基础上鉴定了中国仓鼠卵巢细胞(CHO)细胞系中的16个蛋白点。与已报道的方法比较,本方法蛋白回收率提高到90%左右;与传统的胶内酶解方法比较,本方法具操作简单,实验流程较短;涉及的试剂较胶内酶解少,因此带来的杂质影响小;在蛋白转印以后还可与许多其它实验方法相结合,比如免疫印迹等优点。

An improved method of in-solution digestion of gel-separated proteins for mass spectrometry （MS） identification was developed. Transfer the gel-separated proteins onto the polyvinylidene difluoride （PVDF） membrane; neutralize the solute with NH4HCO3 to the weak alkaline in which the protein was thereafter digested. Interferences arising from the impurities in gel to the MS detection of proteins can thus be eliminated. The method was verified with a standard protein, and a real sample of Chinese Hamster Ovary （CHO） cells was analyzed using the proposed procedures. The protein recovery of the procedures was enhanced up to about 90%. Compared with the traditional in-gel digestion, the present method has advantages of （ 1 ） simplification of operation and the shorten course, （2） less reagent bring less troublesome arising from the impurities, （3） hyphenation with other approaches such as immuno-detection of posttranslational modifications is possible after transferring of proteins.