PEG化rhG-CSF的修饰位点识别研究  

Methods for Identification of Conjugation Site on PEGylated Protein

作　　者：李修乐[1,2] 刘琳[1,2] 高建萍[2] 张竞[2] 孔英俊[2] 王明林[1] 张贵锋[2] 苏志国[2] 

机构地区：[1]山东农业大学食品科学与工程学院,山东泰安271018 [2]中国科学院过程工程研究所生化工程国家重点实验室,北京100190

出　　处：《药物生物技术》2013年第4期348-352,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金项目(No.21076215)

摘　　要：研究聚乙二醇(PEG)修饰的重组粒细胞集落刺激因子(rhG-CSF)上PEG的偶联位点识别方法。采用聚乙二醇单甲醚琥珀酰亚胺碳酸酯(mPEG-SC 5k)修饰rhG-CSF,利用凝胶过滤法对PEG-rhG-CSF进行分离;采用质谱分析结合胰蛋白酶和糜蛋白酶降解法以及氨基酸组成分析法等技术,研究PEG-rhG-CSF上PEG偶联位点识别方法。胰蛋白酶降解法结合质谱分析结果表明PEG-rhG-CSF酶解产物中N末端多肽T1PLGPASSLPQSFLLK16的残留量为4%,多肽C17LEQVR21的残留量为96%,表明96%的修饰位点发生在N末端的苏氨酸上,4%的修饰位点发生在16位的赖氨酸上,实验采用糜蛋白酶降解法和氨基酸组成分析法进一步测定了PEG修饰位点以及不同修饰位点的比例,与基于胰酶降解和质谱分析法获得的偶联位点结果一致。结论:采用多种方法对PEG-rhG-CSF上PEG偶联位点进行了识别。胰蛋白酶降解法无需对不同修饰位点的PEG-rhG-CSF进行分离,通过多肽含量变化可获得识别修饰位点及比例,HPLC分析过程中多肽分离度对定量结果有影响。胰蛋白酶降解法结合糜蛋白酶降解法直接获得PEG偶联位点信息,但糜蛋白酶降解位点较多,影响方法的通用性。反相色谱法或基于氨基酸组成分析的方法操作过程简单,但准确度较低。To investigate the modification site of PEG-modified recombinant granulocyte colony-stimulating factor（PEG-rhG-CSF）,rhG-CSF was modified with methoxy PEG succinimidyl carboxymethyl ester（mPEG-SC,mean MW 5k）.The rhG-CSF and PEG modified rhG-CSF（PEG-rhG-CSF） were separated using gel filtration chromatography.HPLC-MS analysis coupled with trypsin and / or chymotrypsin digestion,amino acid components analysis was applied for identification of PEG-linking site on PEG-rhG-CSF.PEGrhG-CSF was digested by trypsin.Peptides in the digest mixture were analyzed using HPLC-MS.The N-terminal peptide,T1PLGPASSLPQSFLLK16,remaining was 4%,and the C17LEQVR21remainly was 96% compared with that for the digested rhG-CSF,indicating that 96% of PEG was conjugated on the N terminal of rhG-CSF,Thr1,and 4% were conjugated on the Lys16.The PEGlinking peptides in the trypsin digest of PEG-rhG-CSF were separated,and were further digested by chymotrypsin.The PEG-linking site on Thr1and Lys16were confirmed according to the peptides,PQSF and EQVR.The ratio of Thr1to Lys16was 24： 1.Amino acid components of PEG-linking peptides separated from tryptic digestion of PEG-rhG-CSF was analyzed using HPLC.The result was closed to that obtained by the former two methods.Three methods were carried out for identification of PEG-linking site on PEG-rhGCSF.HPLC-MS detection of the peptides in the trypsin digestion may provide the linking-site information.The abundance of peptides in the digest mixture may provide the ratio of different PEG-linking sites.For this method,separation of PEG-rhG-CSF isomers is not necessary.The chymotrypsin digestion of PEG-linking peptides separated from the trypsin digested PEG-rhG-CSF may provide more straightforward information on modification site.This method is not fit for some proteins containing lots of site that can be degraded by chymotrypsin.Amino acid analysis based method is easy to be carried out,but separation of PEG-linking peptides is needed.Amino acid sequence in the PEG-linking peptide might be con

关 键 词：聚乙二醇 修饰 重组粒细胞集落刺激因子 偶联位点 识别 质谱 酶解 

分 类 号：TQ93[化学工程]