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建立了生物质谱研究rhEPO一级结构的系列方法,包括分子量、唾液酸含量以及N-糖含量等糖基化性质的测定方法,并用于4种国产rhEPO样品的分析;在此基础上优化酶切条件,使测定的肽质量指纹谱(PMF)的覆盖率均达到98%,成功鉴定了4个样品中的全部N-糖基化位点以及对分子中两对二硫键进行了结构确证,测定4个样品分子量分别为27754.1±27.4Da、27941.4±23.2Da、27682.5±22.0Da和27914.7±22.5Da;唾液酸含量分别为5.0%、4.8%、5.6%和5.4%;糖含量分别为34.3%、34.7%、34.1%和34·6%。此外还对4种产品在O-连接糖型及N-连接糖型上的区别做了初步探讨。
A series of methods for studying the primary structure of rhEPO by bio-mass spectrometry were established, including the determination of the glycosylation properties such as molecular weight, sialic acid content and N-glycan content. The method was applied to the analysis of four domestic rhEPO samples. On the basis of this, Conditions, so that the measured peptide mass fingerprinting (PMF) coverage reached 98%, successfully identified four samples of all N-glycosylation sites and the structure of the two pairs of disulfide bonds were confirmed, The molecular weights of four samples were 27754.1 ± 27.4Da, 27941.4 ± 23.2Da, 27682.5 ± 22.0Da and 27914.7 ± 22.5Da, respectively. The contents of sialic acid were 5.0%, 4.8%, 5.6% and 5.4%, respectively. The contents of carbohydrate were 34.3% , 34.7%, 34.1% and 34.6% respectively. In addition, the differences between O-linked glycoforms and N-linked glycans were also discussed.