Analysis of Neuro-Oxysterols and Neuro-Sterols
Much of our understanding of the biochemistry of neuro-sterols and neuro-oxysterols depends on their reliable detection and quantification in brain tissue, CSF, plasma and in cells of the nervous systems. Both gas chromatography – mass spectrometry (GC-MS) and liquid chromatography (LC)-MS methods have been extensively applied. Gold standard GC-MS methods are based on the classic paper by Dzeletovic et al describing the analysis of oxysterols in plasma (Dzeletovic, Breuer, Lund & Diczfalusy, 1995; Heverin et al., 2004; Schott & Lutjohann, 2015). There are many LC-MS method, with and without the use of derivatisation. A major study using LC-tandem mass spectrometry (MS/MS) has been performed by Russell and colleagues in Dallas in which they analysed over 3,000 serum samples (Stiles, Kozlitina, Thompson, McDonald, King & Russell, 2014). Their analytical method relied on multiple reaction monitoring (MRM) exploiting a tandem quadrupole to achieve maximum sensitivity and with use of multiple isotope labelled standards for quantification (McDonald, Smith, Stiles & Russell, 2012). To achieve high sensitivity others have exploited derivatisation in combination with LC-MS/MS (Honda et al., 2009; Roberg-Larsen et al., 2014; Sidhu et al., 2015; Xu, Korade, Rosado, Liu, Lamberson & Porter, 2011).
Our preference is to use the Girard P (GP) derivatisation reagent in combination with enzymatic oxidation in a methodology called “enzyme-assisted derivatisation for sterol analysis” EADSA (Crick et al., 2015; Griffiths et al., 2013). The method is applicable to any sterol/oxysterol with an oxo group or with a hydroxy group amenable to enzymatic oxidation to a carbonyl. The most commonly used enzyme is cholesterol oxidase which converts 3β-hydroxy-5-ene sterols to 3-oxo-4-enes and 3β-hydroxy-5α-hydrogen structures to 3-ones (Karu et al., 2007). The oxo group is then reacted with the GP hydrazine reagent to give a GP hydrazone which effectively tags the sterol/oxysterol with a positive charge (Figure 5). The consequence of charge-tagging is a major improvement in LC-MS sensitivity. Besides enhancing signal, the charge-tag directs fragmentation in MS/MS or in MS with multistage-fragmentation (MSn), improving confidence for identification and allowing the identification of unexpected sterols/oxysterols (Abdel-Khalik et al., 2018; Griffiths et al., 2019a; Ogundare et al., 2010). We find the use of MS3 in ion-trap mass spectrometers particularly valuable for structural elucidation, where in a first step the GP-derivative [M]+ ion is selected (MS1), in a second step the [M]+ ion is fragmented (MS2), the major fragmentation route is the loss of pyridine (Py) from the derivative to give a [M-Py]+ ion, which is then fragmented in the MS3 step to reveal structurally informative fragment-ions (Figure 5). To augment MS3 we like to combine fragmentation information with exact mass information available on Fourier transform hybrid and tribrid mass spectrometers. We have extensively utilised this method to identify and quantify neuro-sterols and neuro-oxysterols in brain and in CSF and to monitor the import and export of these molecules into and out of brain (Iuliano et al., 2015). We have now extended this method to on-surface analysis of tissue slices to localise neuro-oxysterols and neurosterols in mouse brain (Yutuc et al., 2020).