应用基质辅助激光解析电离飞行时间质谱和N端测序鉴定重组人粒/巨噬细胞集落刺激因子(rhGM-CSF)的结构

Characterization of rhGM-CSF Structure by MALDI-TOF-MS and N-terminal Sequencing

目的 :应用基质辅助激光解析电离飞行时间质谱 (MALDI -TOF -MS)和N端测序方法鉴定重组人粒 /巨噬细胞集落刺激因子 (rhGM -CSF)的结构。方法 :1 用Edman降解法进行N端测序。 2 应用MALDI-TOF -MS测定分子量并鉴定纯度。 3 通过还原、烷基化确定rhGM -CSF分子中二硫键数目。 4 通过蛋白内切酶Glu -C酶切的肽质量指纹谱确证氨基酸序列并确证其中两对二硫键的配对。结果 :1 测定的rhGM -CSFN端 15个氨基酸序列与从cDNA推导的序列一致。 2 测定的rhGM -CSF分子量与理论计算值一致。 3 证明rhGM -CSF没有自由巯基 ,含有两对二硫键 ,与理论值一致。 4 证明rhGM -CSF的氨基酸序列是正确的 ,并确证其中的两对二硫键配对正确。结论 :确证重组人粒 /巨噬细胞集落刺激因子 (rhGM -CSF)的结构是正确的 ,没有修饰、变异和氨基酸的丢失。应用的方法灵敏、准确。

Objective:Confirmation of rhGM-CSF structure by MALDI-TOF-MS and N-terminal sequencing.Methods:1.To measure N-terminal sequence of rhGM-CSF by Edman degradation reaction.2.To measure the molecular weight of rhGM-CSF by MALDI-TOF-MS.3.To determine the number of disulfide bond by comparison molecular weight of rhGM-CSF with reduced and alkylated rhGM-CSF.4.To confirm the amino acid sequence of rhGM-CSF and the location of disulfide bonds by peptides mass fingerprinting.Results:1.N-terminal sequence of rhGM-CSF measured are identical with the sequence deduced from cDNA sequence.2.The measured molecular weight of rhGM-CSF is identical with the calculated value.3.There are two disulfide bonds in rhGM-CSF.4.The amino acid sequence of rhGM-CSF and the location of disulfide bonds are correct.Conclusion:rhGM-CSF structure is correct,there are no modification,mutation and amino acid deletion in the expression,refloding and purification of rhGM-CSF.Applied methods is sensitivity,accurate and reliable.