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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、可靠
OBJECTIVE: To identify the structure of recombinant human granulocyte / macrophage colony stimulating factor (rhGM-CSF) using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing. Methods: 1 N-terminal sequencing was performed by Edman degradation. 2 The molecular weight was determined using MALDI-TOF-MS and the purity was determined. 3 Determine the number of disulfide bonds in the rhGM-CSF molecule by reduction and alkylation. 4 The amino acid sequence was confirmed by peptide mass fingerprinting of the Glu-C endonuclease and the pairing of two pairs of disulfide bonds was confirmed. Results: 1 The 15 amino acid sequence of rhGM-CSFN was consistent with the deduced sequence from cDNA. 2 measured rhGM -CSF molecular weight consistent with the theoretical calculations. 3 Proof that rhGM-CSF has no free thiols and contains two pairs of disulfide bonds, consistent with the theoretical value. 4 Proved that the amino acid sequence of rhGM-CSF was correct and confirmed that two pairs of disulfide bonds were correctly paired. Conclusion: The structure of recombinant human granulocyte / macrophage colony stimulating factor (rhGM-CSF) is confirmed to be correct without modification, mutation and amino acid loss. The method used is sensitive, accurate and reliable