重组人粒细胞巨噬细胞集落刺激因子质量肽图分析及二硫键定位

Peptide Mass Mapping Analysis and Localization of Disulfide Bonds of Recombinant Human Granulocyte- macrophage Colony Stimulating Factor

关键词:  人粒细胞巨噬细胞集落刺激因子  质量肽图  二硫键  液相色谱-质谱联用

Key words:  human granulocyte-macrophage colony stimulating factor  peptide mass mapping  disulfide bonds  LC-MS/MS

摘要:

目的 采用液相色谱-质谱联用技术绘制重组人粒细胞巨噬细胞集落刺激因子的质量肽图并鉴定二硫键位点。方法 重组人粒细胞巨噬细胞集落刺激因子原液经变性、还原、烷基化、脱盐、胰蛋白酶和V₈酶分别酶解后进行质量肽图分析；原液经变性、烷基化、脱盐、V₈酶和V₈酶结合胰蛋白酶分别酶解后进行二硫键定位鉴定。结果 胰蛋白酶酶切的质量肽图共发现14个匹配肽段，V₈酶酶切的质量肽图共发现18个匹配肽段，肽段覆盖率均为100%。经V₈酶酶切仅鉴定到1种二硫键匹配方式即Cys55-Cys97，经V₈酶结合胰蛋白酶酶切鉴定到2种二硫键匹配方式即Cys55-Cys97和Cys89-Cys122。结论 应用液相色谱-质谱联用技术进行质量肽图分析和二硫键定位可作为重组蛋白药物质量控制方法。

Abstract:

OBJECTIVE To analyze the peptide mass mapping and localization of disulfide bonds of recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF) by using mass spectrometry combined with ultra-performance liquid chromatography. METHODS The bulk of rhGM-CSF was denatured, reduced, alkylated and finally digested by the enzyme of trypsin and V₈ respectively to analyse the peptide mass mapping. The bulk was denatured, alkylated and digested by V₈ and V₈ with trypsin respectively to identify the sites of disulfide bonds. RESULTS Fourteen matched peptides were found in the peptide mass mapping of rhGM-CSF digested by trypsin and eighteen matched peptides were found digested by V₈. The fraction of coverage was both 100.0%. The site of disulfide bond Cys55-Cys97 was identified by digestion of V₈, and the types of disulfide bonds Cys55-Cys97 and Cys89-Cys122 were identified by digestion of V₈ with trypsin. CONCLUSION The peptide mass mapping analysis and localization of disulfide bonds using LC-MS/MS can be used for the quality control of recombination protein.