rhIL-12二硫键、N-糖基化位点及C端氨基酸序列分析

Analysis of rhIL-12 Disulfide Bond And N-glycosylation Sites and C-terminal Amino Acid Sequence

重组人白细胞介素12(rhIL-12)是一种已经用于治疗肿瘤,寄生虫、病毒性感染及造血障碍等疾病研究的异二聚体糖蛋白。结构确证是质量控制的重要内容,此研究对CHO细胞表达的rhIL-12二硫键配对方式、N-糖基化位点以及C端氨基酸序列进行了分析,使用Trypsin、Chymotrypsin和Glu-C三种酶分别对rhIL-12进行非还原酶解,尽可能地在其所有半胱氨酸残基之间断裂而形成二硫键相连的肽段,然后使用LC-MS/MS对酶解后的肽段样品进行分析,确定了rhIL-12样品中存在和理论配对方式相符的7对二硫键。将rhIL-12二硫键还原后并烷基化修饰保护,分别采用Trypsin,Chymotrypsin和GluC进行酶解,并用LC-MS/MS对酶解后肽段进行了质谱肽图及C端氨基酸序列分析,确定了rhIL-12 p35亚基C端氨基酸序列的8个氨基酸、p40亚基C端氨基酸序列的15个氨基酸。对rhIL-12样品还原及烷基化后用Trypsin变性酶解,所得肽段在H2O及H218O水中分别用PNGase F糖苷酶处理酶切产物。并通过二级质谱分析脱糖后糖肽段分子量变化,从而确定了rhIL-12的3个N糖基化修饰位点,分别为p35亚基的71位和85位以及p40亚基的200位。通过建立酶解结合二级质谱鉴定的方法,证明了新药rhIL-12的二硫键位点、C端氨基酸序列和糖基化位点与理论一致。

Recombinant human interleukin- 12 （rhIL-12） is a heterodimeric glycoprotein that has been used to treat diseases such as tumor, parasites, viral infections and hematopoietic disorders. For the structure confirmation is important for quality control, in this paper, the disulfide linkage, N-glycosylation sites and C-terminal amino acid sequences of rhIL-12 expressed by CHO cells were analyzed, and the rhIL-12 underwent enzymolysis using three non-reducing enzymes： Trypsin, Chymotrypsin and Glu-C, to break between cysteine residues and form the disulfide-linked peptides, and then peptide samples were analyzed using LC-MS/MS to identify seven pairs of disulfide bonds presenting in rhIL-12 sample that match the theoretical conditions. After reduction of disulfide bond and alkylation modification protection, the rhIL-12 underwent enzymolysis using Trypsin, Chymotrypsin and GluC, and mass spectrum peptide mapping and C-terminal amino acid sequence analysis of the peptide segments were then carried out using LC-MS/MS, to identify eight amino acids at p35 subunit C-terminal and 15 amino acids at p40subunit C-terminal of rhIL-12. After reduction and alkylation, the rhIL-12 samples were degenerated and enzymolysised, and the enzyme-digested products of peptide segments were thereafter treated with PNGase F in H2O and H218O respectively. Through tandem mass spectrometry analysis of the change in molecular weight of peptide fragments, three N glycosylation sites of rhIL-12 were exactly identified, which were site 71 and site85 of p35 subunit, and site 200 of p40 subunit. Through establishing the method combining enzymolysis with mass spectrometry identification, ultimately it was demonstrated that disulfide bond site, C-terminal amino acid sequence and glycosylation sites of new drug rhIL-12 were consistent with the theoretical conditions.