目的:建立新型定点修饰的聚乙二醇化重组人生长激素(PEG-rhGH)修饰位点的研究方法,为该类新型修饰技术产物的质量评价提供依据。方法:采用MALDI-TOF质谱分析修饰产物中聚乙二醇(PEG)的修饰个数及修饰肽段的质量数;采用LC-Q-TOF质谱分析修饰位点,分析色谱柱为UPLC色谱柱(Protein ACQUITY BEH C4 Column,150 mm×2.1 mm,1.7 μm,300Å),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,进行梯度洗脱,柱温为40 ℃。质谱数据采集条件为MSE模式,一级质谱能量4 eV,二级碎裂电压30~55 eV。结果:修饰蛋白中PEG的平均相对分子质量为30 000,并且每个PEG-rhGH分子中仅存在1个PEG修饰;原型蛋白重组人生长激素中第134位精氨酸被替换为赖氨酸,且该赖氨酸的ε-氨基与连接子HOOC-O-CH2-CH2-N3中的羧基端共价结合,连接子另一端的叠氮基团与活化后的PEG偶联后生成修饰产物。结论:联合运用多种质谱技术,通过比对人生长激素国家对照品、原型蛋白与修饰蛋白的酶切肽图谱及对特定肽段的一级、二级碎片的分子量分析,可以确定新型定点修饰的PEG-rhGH的修饰位点。
Objective: To establish a new site-specific modified pegylated recombinant human growth hormone(PEG-rhGH) modification site research method, and provide a basis for the quality evaluation of the products of this new type of modification technology. Methods: MALDI-TOF mass spectrometry was used to analyze the number of modified PEGs and the mass of modified peptides. LC-Q-TOF mass spectrometry was used to analyze the modification sites and the analysis column was UPLC column(ACQUITY UPLC Protein BEH C4, 150 mm×2.1 mm, 1.7 μm, 300Å). Gradient elution was performed using 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution. The column temperature was set to 40 ℃. The conditions of mass spectrum were a below: MSE mode with a primary mass spectrum energy of 4 eV and a secondary fragmentation voltage of 30-55 eV. Results: The average relative molecular mass of PEG in the modified protein was 30 000, and only single PEG molecule was found in PEG-rhGH molecule; the arginine at position 134 in the recombinant human growth hormone of the prototype protein was replaced with lysine, and the ε-amino group of the lysine was covalently linked to the carboxyl end of the linker HOOC-O-CH2-CH2-N3, and the azide group at the other end of the linker was coupled with the activated PEG to generate a modified product. Conclusion: Combined with a variety of mass spectrometry techniques, by comparing the national reference substance of human growth hormone, the cleavage peptide map of the prototype protein and the modified protein, and the molecular weight analysis of the primary and secondary fragments of specific peptides, it is capable of determining the new site-specific modified poly modification sites of PEG-rhGH.
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