溴化氰裂解结合电喷雾串联质谱测定电泳分离蛋白的C端序列

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。

C-Terminal Sequencing for Recombinant Proteins by Tandem Mass Spectrometry Combined with Cynogen Bromide Cleavage

A new method based on ESI-MS/MS technique combined wit h cyanogen bromide cleavage was developed to recombinant human-tumor necros is factor receptor(rhTNFR)and Neuregulin.At first,a theoretical cyanogen bro mide cleavage was performed according to the protein sequence.If the molecular weight of C-terminal peptide is between 500 and 5000 U,then SDS-PAGE sepa ration and in gel cyanogen bromide cleavage was performed.At last,the molecula r weight of cleavaged peptides was measured by MALDI-TOF-MS and the C-ter minal 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.T hese results show that the new method is sensitive,effective and can be widely used in the quality control of some recombinant protein drugs and characterizati on of the primary structures of proteins and peptides.