重组人促红细胞生成素的HPLC/ESI/MS肽图谱分析

HPLC/ESI/MS Analysis of rHuEPO

重组人促红细胞生成素（rHuEPO）是一种复杂的糖蛋白药物，含有三个氮连接和一个氧连接的糖基化位点。肽图谱分析是糖蛋白分析的有效手段，可是由于糖基化的影响，不是所有理论酶解位点都酶解了，需要实验验证。本文采用谷氨酸内切酶(Glu-C)将rHuEPO所有糖基化位点酶解成含糖肽段，HPLC/ESI/MS肽图谱分析，并通过源内碰撞诱导解离（In-source CID）分析了氨基酸序列。理论肽段有7段，检出了6段，只有一个三肽未检出。实验得到了含双硫键的肽段，通过CID分析确证了小分子肽段的氨基酸序列。

Recombinant human erythropoietin (rHuEPO) is highly glycosylated. It has three N-linked and one O-linked glycosylation sites. The analytical strategy includes enzymatic digestion, HPLC separation and structural determination. However with the influence of glycosylation and disulfide bonds, the enzymatic cleav age sites were unpredictable. HPLC/ESI/MS peptide mapping is a powerful tool for the analysis of rHuEPO. rHuEPO was digested with Glu-C and separated by microbore HPLC, and analyzed by online ESI/CID/MS. All the peptides and glycopeptides except a tripeptide were identified. Two peptides linked through disulfide bond were i dentified by using in-source CID; peptide amino acid sequence was also analyzed by this technique.