Crick Peter J, Aponte Jennifer, Bentley T William, Matthews Ian, Wang Yuqin, Griffiths William J
Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
Biochem Biophys Res Commun. 2014 Apr 11;446(3):756-61. doi: 10.1016/j.bbrc.2014.01.173. Epub 2014 Feb 10.
Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MS(n) fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.
氧化甾醇是胆固醇的氧化形式，是胆汁酸和类固醇激素合成过程中的中间体。它们也是核受体和G蛋白偶联受体的配体。由于氧化甾醇在生物系统中的丰度较低，加上缺乏强发色团且在质谱（MS）中电离特性较差，因此对其进行分析具有挑战性。我们之前使用酶辅助衍生化甾醇分析（EADSA）来鉴定和定量生物样品中的氧化甾醇。该技术依靠用吉拉德P试剂标记甾醇以引入带电荷的季铵基团。在此，我们比较了几种改良的类吉拉德试剂，并表明永久电荷对于高效的MS(n)碎片化至关重要。然而，我们发现该试剂可以扩展到包括潜在稳定同位素标记的位点，而不会损失性能。
