摘要
C端测序是蛋白质及多肽一级结构确认的重要组成部分,也是重组蛋白药物质量控制的重要依据。建立了溴化氰裂解结合电喷雾串联质谱测定蛋白质C端序列的方法,并应用于重组人肿瘤坏死因子受体和纽兰格林的C端测序。首先根据待测蛋白序列进行溴化氰理论裂解,如果C-端肽段理论分子量在500~5000U之间,则将待测样品进行SDS-PAGE分离,考马斯亮兰染色,然后进行胶内溴化氰裂解,最后应用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）测定C-端肽段的分子量,电喷雾串联质谱对C端肽段进行测序。应用本方法分别测定了这两个蛋白质C端19个和11个氨基酸残基序列。研究结果表明：本方法灵敏、有效、实用性较强,可适用于部分重组蛋白药物的质量控制和蛋白质的结构确证,是对目前蛋白质C端测序方法的有效补充。
A new method based on ESI-MS/MS technique combined with cyanogen developed to recombinant human-tumor necrosis factor receptor （rhTNFR） and Neuregul bromide cleavage was in. At first, a theoretical cyanogen bromide cleavage was performed according to the protein sequence. If the molecular weight of Cterminal peptide is between 500 and 5000 U, then SDS-PAGE separation and in gel cyanogen bromide cleavage was performed. At last, the molecular weight of cleavaged peptides was measured by MALDI-TOF-MS and the C-terminal sequence was obtained by nano ESI-MS/MS. 19 C-terminal amino acid sequences of rhTNFR and 11 of rhNeuregulin were determined by this method. These results show that the new method is sensitive, effective and can be widely used in the quality control of some recombinant protein drugs and characterization of the primary structures of proteins and peptides.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2008年第4期514-517,共4页
Chinese Journal of Analytical Chemistry
基金
北京市自然科学基金资助项目(No.2062022)
关键词
C端测序
溴化氰裂解
电喷雾串联质谱
重组蛋白
C-terminal sequencing, cyanogen bromide cleavage, electrospray ionization tandem mass spec-trometry, recombinant human tumor necrosis factor receptor and Neuregulin
