副猪嗜血杆菌脂酰辅酶A合成酶活性质谱定性分析

Mass Spectrum Analysis of Acyl-CoA Synthetase Activity of Haemophilus parasuis

利用已纯化的FadD1和FadD2进行体外重构生化反应,通过质谱分析技术对2个脂酰辅酶A合成酶进行活性定性分析。体外酶活反应产物经UPLC-MS/MS质谱分析显示,全扫描模式,中性丢失扫描模式或多反应监测模式均显示酶活反应产物有脂酰辅酶A的生成,而且FadD1对脂酰辅酶A的转化效率要明显高于FadD2。质谱分析数据进一步确认脂酰辅酶A合成酶的生化功能,表明其在HPS存活过程中发挥着重要的生理功能,是其致病的关键蛋白之一。成功建立了脂酰辅酶A合成酶体外质谱分析方法,可为病原细菌相关基因功能研究提供技术基础。

In this study, purified FadD1 and FadD2 were used for reconstruction of biochemical reactions in vitro and the activity of two acyl-CoA synthetases was qualitatively analyzed by mass spectrometry.Survey mode, neutral loss scan mode and multi-reaction monitoring mode all showed that products from enzyme activity reaction in vitro contained acyl-CoA confirmed by UPLC-MS/MS,meanwhile the conversion efficiency of FadD1 to acyl-CoA was significantly higher than FadD2.Mass spectrum data present further confirmed the biochemical function of acyl-CoA synthetase, indicating that it plays a key physiological role in HPS survival and is one of the key proteins in its pathogenicity.Additionally, the in vitro mass spectrometric analysis method of acyl-CoA synthetase was successfully established, which provides a technical basis for the study of the function of related genes in pathogenic bacteria.