Hybrid 213 nm photodissociation of cationized Sterol lipid ions yield [M]^+. Radical products for improved structural characterization using multistage tandem mass spectrometry

Highlights

• Hybrid UV photodissociation and multistage tandem mass spectrometry is a novel analysis strategy for sterol lipids.

• 213 nm UVPD-MS/MS of cholesterol and oxysterols produces abundant [M]^+. radical cation product ions.

• HCD-MS³ of these products enables unambiguously structural assignment, including for differentiation of isomeric oxysterols.

• HCD-MS/MS and UVPD-MS³ enables detailed structural assignment of cholesterol esters, including site(s) of unsaturation.

Abstract

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., MSⁿ) 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 MS³ 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-MS³ approach is shown to enable assignment of the sterol backbone, and localization of the site(s) of unsaturation within esterified fatty acyl chains.

Introduction

Sterols are a class of lipid whose members include cholesterol, oxysterols and sterol esters. Cholesterol plays extremely important roles in mammalian systems, by providing fluidity to cellular membranes, as the precursor to derivatives including oxysterols, steroid hormones and bile acids and acting as a ligand for receptors [1]. Oxysterols are oxidised derivatives of cholesterol [2], whose most well-known role is as early intermediates in the synthesis of bile acids. Oxysterols have also been shown to be important to many different cellular processes, as ligands for several receptor classes including nuclear receptors [3], and GPCR’s [4]. 25-hydroxycholesterol is believed to play an important role in the activity of the immune system [5]. Changes in oxysterol abundance have also been observed in several diseases including neurodegeneration [6], atherosclerosis [7] and inflammatory diseases. As oxysterols interact with a variety of enzymes, including many within the cytochrome family, they are a useful endogenous analyte for detection of metabolic disorders. For example, 4β-hydroxycholesterol is used to monitor CYP3A4/5 activity [8]. Sterols can be esterified with fatty acids to form sterol esters, which are involved in the transport and storage of sterols around the body [9]. Sterol esters play an important role in maintaining sterol homeostasis, via packing into lipid droplets for cellular storage. Oxysterol esters are known to interact with a range of enzymes, and disruptions in their pathways have been implicated in diseases, including atherosclerosis.

The ‘gold standard’ methods used for analysis of sterols are gas chromatography-mass spectrometry (GC-MS) [10,11], and high performance liquid Chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) [12,13]. GC-MS typically involves analysis of sterol lipids as their trimethyl silyl ethers. The use of electron ionization (EI) for analyte ionization prior to MS analysis results in extensive fragmentation of the M^+. radical molecular cation, providing detailed structural information for differentiation of isomeric sterol lipid species, particularly when combined with the characteristic GC retention time determined from authentic standards. Limitations to the universal application of GC-MS methods, however, include the requirement to first hydrolyse conjugates (e.g., cholesterol esters) under alkaline conditions that results in an inability to characterize them as their intact molecular structures.

In contrast, when using HPLC-ESI-MS methods, alkaline hydrolysis is not necessary, allowing for conjugated species to be directly introduced and subsequently analyzed. Detection using ultra-high resolution accurate mass spectrometry (UHRAMS) enables the determination of elemental composition; however, it does not provide any information about the structure. Therefore, tandem mass spectrometry (MS/MS) is typically required to provide additional structural information for unambiguous sterol lipid identification. However, conventional collision induced dissociation (CID) methods typically used for ion activation in MS/MS based analysis strategies often fail to provide sufficient structural information (the dominant channel typically involve loss of water) to differentiate between isomeric sterol lipid species. As a consequence, the success of HPLC-ESI-MS/MS methods are heavily dependent on the efficiency of the chromatographic separation method, to enable matching of endogenous lipids with the characteristic retention times of their reference standards.

An additional challenge associated with the analysis of sterols is that they generally exhibit relatively poor ionization efficiency when using ESI methods. Therefore, derivatization methods, combined with extraction protocols optimized for sterol enrichment, are commonly employed in LC-MS workflows to increase sterol ionization efficiency. Griffiths et al. has reviewed these derivatization methods [13], with the most popular being the Girard P hydrazine reagent, that results in the introduction of a quaternary amine as a permanent charge to sterol species containing a native oxo group, or following initial cholesterol oxidase conversion of the 3-hydroxy group found on many sterols to their 3-oxo forms [[14], [15], [16], [17], [18]]. Another benefit of derivatization is that the fragmentation observed using multistage CID-MS/MS (i.e., -MSⁿ) methods results in the formation of novel structurally informative product ions for confirmation of known sterols, and to identify and characterize new sterol species [19]. A potential limitation to this derivatization approach however, is that it requires the presence of a 3-oxo or 3-hydroxy group on the sterol, thereby making it unsuitable for the direct analysis of conjugated species such as cholesterol esters.

As an alternative to well established CID (or higher-energy collision induced dissociation (HCD)) ion activation methods for the MS/MS based analysis of cholesterol, oxysterols and cholesteryl esters [[20], [21], [22], [23], [24]], 193 nm or 213 nm ultraviolet photodissociation (UVPD) is an emerging ion activation technique for MS/MS-based biomolecular analysis [25], including for lipidomics. To date, UVPD-MS/MS, or hybrid UVPD- and/or CID -MSⁿ methods, have been demonstrated to yield novel product ions that enable the improved structural characterization of multiple lipid classes, including Lipid A [26,27], glycerophospholipids [[28], [29], [30], [31], [32]], sphingolipids [33,34], and unsaturated fatty acids [35,36], with particular applicability for assigning the sites of CC double bond locations, and/or sn-linkage positions within these molecular species. To date, however, the potential for UVPD-MS/MS, or sequential UVPD and CID or HCD-MSⁿ methods, to provide novel structural information for the improved identification and characterization of sterol lipids, including cholesterol, oxysterol and cholesteryl esters, has not been reported.