亲和层析结合生物质谱技术分析鉴定猕猴血清中的重组人内皮抑制素  

Extraction and Characterization of Rh-Endostatin in Rhesus Serum by Affinity Chromatography Combined with Peptide Mass Fingerprinting

作　　者：刘尚义[1] 刘秀文[1] 蔡耘[1] 宋海峰[1] 应万涛[1] 朱宝珍[1] 李蕾[1] 汤仲明[1] 钱小红[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《分析化学》2003年第3期277-282,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金重点项目资助课题 (No .39930 180 )

摘　　要：建立了将固相亲和层析与生物质谱技术相结合 ,分离提取鉴定给药猕猴血清中含有 6×His序列的重组人内皮抑制素的方法。用固相免疫亲和层析和金属螯合层析两种方法分别提取并富集血清中的重组药物 ,采用基质辅助激光解吸 电离飞行时间质谱的直接分析和肽质量指纹谱分析与数据库检索 ,分别对两种亲和提取方法得到的药物蛋白前体collagenXVIII[Homosapiens](IDofSWISS -PROT TrEMBL :Q8WXI5 )It is difficult to analyze protein drug in biological sample since the concentration of protein drug in it is very low,and lots of impurities with similar structure as protein drug will disturb the analysis. In this research, a method combining immobilized affinity chromatography with biological mass spectrometry to extract and characterize recombined protein drug from animal serum was developed. Protein drug was extracted and enriched from rhesus serum after i.v. 13.5 mg/kg rh-endostatin 2 min and 20 min with two affinity chromatography methods, immobilized immunoaffinity chromatography (IAC) and immobilized metal ion affinity chromatography (MAC). The mass information of protein drug, metabolites and drug-protein complexes in the gel was acquired by matriv-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of affinity gel directly. Protein drug in the affinity gel was digested with trypsin and the masses of generated peptides were measured with MALDI-TOF-MS. The data obtained from peptide mass fingerprinting (PMF) was searched in protein database, and collagen XVIII [Homo sapiens] (ID of SWISS-PROT/TrEMBL:Q8WX15), a prototype of endostatin in vivo was identified.

关 键 词：分析 鉴定 猕猴 血清 人内皮抑制素 亲和层析 生物质谱 肽质量指纹谱分析 蛋白质药物 代谢 基因重组 分析 

分 类 号：O657.63[理学—分析化学]