温度和pH对重组人血管内皮抑制素脱酰胺的影响

Effect of temperature and pH on the deamidation process of Asn in rhEndostain

目的利用液质联用技术研究温度和pH对重组人血管内皮抑制素中天冬酰胺(Asn)脱酰胺过程的影响。方法在不同的温度和pH值条件下,对重组人血管内皮抑制素样品溶液进行孵化,酶解处理后利用液质联用技术识别发生脱酰胺的多肽并测定其含量随样品放置温度和pH条件的变化。结果质谱分析结果表明酶解产物中多肽SVWHGSDPNGR序列中的Asn发生了脱酰胺化,Asn残基生成了天冬氨酸和异天冬氨酸残基,其他位置的Asn均未检测出明显的脱酰胺化;实验获得了不同温度和pH条件下重组人血管内皮抑制素中Asn脱酰胺化的速率及动力学常数;Asn脱酰胺过程随着温度和溶液pH值的升高,Asn脱酰胺速率上升。结论重组人血管内皮抑制素中天冬酰胺的脱酰胺过程受储存温度和pH值影响,研究药用蛋白质在不同条件下的脱酰胺过程对于筛选合适的储存条件、减少活性损失具有重要意义。

Objective To investigate the effect of temperature and pH on the deamidation process of asparaginyl（Asn）in the rhEndostatin using liquid chromatography-tandem mass spectrometry. Methods The samples of rhEndostatin were incubated at different temperature and pH,and then digested by trypsin.The peptides in the digest mixture were analyzed to identify the deamidation site of Asn.The ratio of deamidation of sample inculated at different temperature and pH was determined. Results The Asn in the peptide,SVWHGSDPNGR,was found to deamidate and transform into asparate（Asp）and isoaspartate （isoAsp）.No obvious deamidation in Asn except in the SVWHGSDPNGR was observed.The effect of incubation temperature and pH on the deamidation process and kinetic constant was investigated.The kinetic constant of Asn deamidation is accelerating with the increasing of temperature and the pH of solution. Conclusions The deamidation process of Asn in rhEndostatin is affected by the pH of solution and the temperature of storage.Thus, studying of the deamidation of pharmaceutical proteins in different conditions is important for selecting proper storage environment and reducing the loss of activity.