重组人粒细胞巨噬细胞集落刺激因子（GM-CSF）抗性核心肽的分离与鉴定；通过质谱法确认GM-CSF氨基酸序列
Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.
作者信息
Tsarbopoulos A, Pramanik B N, Labdon J E, Reichert P, Gitlin G, Patel S, Sardana V, Nagabhushan T L, Trotta P P
机构信息
Shering-Plough Research Institute, Kenilworth, New Jersey 07033.
出版信息
Protein Sci. 1993 Nov;2(11):1948-58. doi: 10.1002/pro.5560021116.
A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.
通过高能Cs⁺液体二次离子（LSI）质谱分析对重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）的胰蛋白酶抗性核心肽进行了分离和分析。该分析成功检测到了高质量的二硫键连接的核心肽，并提供了确认二硫键配对存在的信息。同样，对从rhGM-CSF的金黄色葡萄球菌V8蛋白酶消化物中色谱分离得到的肽片段进行LSI质谱分析，快速确认了cDNA衍生序列，并确定了半胱氨酸残基54 - 96和88 - 121之间存在的二硫键。采用电喷雾电离质谱法测量完整蛋白质的分子量，并通过对用β-巯基乙醇还原前后的蛋白质进行比较分析来确定蛋白质分子中二硫键的数量。
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