Objective: To establish a method for disulfide bonding determination of erythropoiesis stimulating pro-tein(ESP). Methods: ESP, removal of sugar chain by peptide-N-glycosidase, was reduced and alkylated, then digested by trypsion. By comparing before and after reductive alkylation peptide mass fingerprinting, to find different peptides in the molecular ion peak [M+H]+ and determine the disulfide bond linking mode. Results: Our results demonstrated there were two disulfide bond in the ESP. One is disulfide Cys'-Cys1" and another is CysB-Cysn. Conclusion: The method to determinate disulfide bonding was successfully established, which combined digest method and mass spectrometry analysis and would be used for quality control for recombinant protein.%目的:建立红细胞生长刺激蛋白(ESP)的二硫键连接方式的测定方法.方法:先将红细胞生长刺激蛋白的糖链用糖苷酶切除,再分别对ESP和还原烷基化后ESP用胰蛋白酶进行酶切,然后用MALDI-TOF测得该蛋白质的肽质量指纹图谱,通过比较还原烷基化前后各肽质量指纹图谱,找到差异肽段的分子离子峰[M+H]+,通过比对该蛋白理论酶切肽的[M+H]+,确定二硫键的连接方式.结果:ESP有4个半胱氨酸,通过比较还原烷基化前后的肽质量指纹图谱定位二硫键的位置为Cys7-Cys161和Cys29-Cys33.与理论上的二硫键连接相符.结论:建立了酶切结合质谱法测定蛋白质二硫键定位的方法,为今后生物技术产品的二硫键连接方式的质量控制提供了有效的方法.
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