West Henry, Reid Gavin E
School of Chemistry, The University of Melbourne, Parkville, Victoria, 3010, Australia.
School of Chemistry, The University of Melbourne, Parkville, Victoria, 3010, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia; Bio 21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010, Australia.
Anal Chim Acta. 2021 Jan 2;1141:100-109. doi: 10.1016/j.aca.2020.10.013. Epub 2020 Oct 21.
Sterols are a class of lipid molecules that include cholesterol, oxysterols, and sterol esters. Sterol lipids play critical functional roles in mammalian biology, including the dynamic regulation of cell membrane fluidity, as precursors for the synthesis of bile acids, steroid hormones and vitamin D, as regulators of gene expression in lipid metabolism, and for cholesterol transport and storage. The most common method employed for sterol analysis is high performance liquid chromatography coupled with tandem mass spectrometry (MS/MS). However, conventional collision induced dissociation (CID) methods used for ion activation during MS/MS typically fail to provide sufficient structural information for unambiguous assignment of sterol species based on their fragmentation behaviour alone. This places a significant burden on the efficiency of the chromatographic separation methods for the effective separation of isomeric sterols. Here, toward developing an improved analysis strategy for sterol lipids, we have explored the novel use of 213 nm photodissociation MS/MS and hybrid multistage-MS/MS (i.e., MS) data acquisition approaches for the improved structural characterization of cholesterol, representative isomeric oxysterols, and cholesteryl esters. Most notably, UVPD-MS/MS of ammoniated, lithiated and sodiated adducts of cholesterol, several representative oxysterol species, and an oxosterol lipid, are shown to give rise to abundant [M] radical cation products, that subsequently fragment during collision induced MS to yield extensive structurally informative product ions, similar to those observed by Electron Ionization, and that enable their unambiguously assignment, including isomeric differentiation of oxysterols. For cholesterol esters, a reversed hybrid collision induced-MS/MS and UVPD-MS approach is shown to enable assignment of the sterol backbone, and localization of the site(s) of unsaturation within esterified fatty acyl chains.
甾醇是一类脂质分子，包括胆固醇、氧化甾醇和甾醇酯。甾醇脂质在哺乳动物生物学中发挥着关键的功能作用，包括动态调节细胞膜流动性、作为胆汁酸、类固醇激素和维生素D合成的前体、作为脂质代谢中基因表达的调节剂以及用于胆固醇的运输和储存。甾醇分析最常用的方法是高效液相色谱与串联质谱（MS/MS）联用。然而，在MS/MS过程中用于离子活化的传统碰撞诱导解离（CID）方法通常无法仅根据甾醇种类的碎裂行为提供足够的结构信息以进行明确的鉴定。这给有效分离异构甾醇的色谱分离方法的效率带来了巨大负担。在此，为了开发一种改进的甾醇脂质分析策略，我们探索了213 nm光解离MS/MS和混合多级MS/MS（即MSn）数据采集方法在胆固醇、代表性异构氧化甾醇和胆固醇酯的结构表征改进方面的新用途。最值得注意的是，胆固醇、几种代表性氧化甾醇种类和一种氧化甾醇脂质的氨化、锂化和钠化加合物的UVPD-MS/MS显示会产生大量的[M]自由基阳离子产物，这些产物随后在碰撞诱导MS过程中碎裂，产生大量具有结构信息的产物离子，类似于电子电离所观察到的，并且能够对它们进行明确的鉴定，包括氧化甾醇的异构体区分。对于胆固醇酯，一种反向混合碰撞诱导-MS/MS和UVPD-MS方法显示能够鉴定甾醇主链，并确定酯化脂肪酰链内不饱和位点的位置。
