Metabolite identification using an ion mobility enhanced data-independent acquisition strategy and automated data processing.

RATIONALE: Liquid chromatography/mass spectrometry is an essential tool for efficient and reliable quantitative and qualitative analysis and underpins much of contemporary drug metabolism and pharmacokinetics. Data-independent acquisition methods such as MSE have reduced the potential to miss metabolites, but do not formally generate quadrupole-resolved product ion spectra. The addition of ion mobility separation to these approaches, for example, in High-Definition MSE (HDMSE ) has the potential to reduce the time needed to set up an experiment and maximize the chance that all metabolites present can be resolved and characterized. We compared High-Definition Data-Dependent Acquisition (HD-DDA), MSE and HDMSE approaches using automated software processing with Mass-MetaSite and WebMetabase. METHODS: Metabolite identification was performed on incubations of glucagon-like peptide-1 (7-37) (GLP-1) and verapamil hydrochloride. The HD-DDA, MSE and HDMSE experiments were conducted on a Waters ACQUITY UPLC I-Class LC system with a VION IMS quadrupole time-of-flight (QTOF) mass spectrometer operating under UNIFI control. All acquired data were processed using MassMetaSite able to read data from UNIFI 1.9.4. WebMetabase was used to review the detected chromatographic peaks and the spectral data interpretations. RESULTS: A comparison of outcomes obtained for MSE and HDMSE data demonstrated that the same structures were proposed for metabolites of both verapamil and GLP-1. The ratio of structurally matched to mismatched product ions found by MassMetaSite was slightly greater for HDMSE than for MSE , and HD-DDA, thus improving confidence in the structures proposed through the addition of ion mobility based data acquisitions. CONCLUSIONS: HDMSE data acquisition is an effective approach for the elucidation of metabolite structures for both small molecules and peptides, with excellent accuracy and quality, requiring minimal tailoring for the compound under investigation.

High-Pressure Ozone-Induced Dissociation for Lipid Structure Elucidation on Fast Chromatographic Timescales.

Ozone-induced dissociation (OzID) is a novel ion activation technology that exploits the gas-phase reaction between mass-selected ions and ozone inside a mass spectrometer to assign sites of unsaturation in complex lipids. Since it was first demonstrated [ Thomas et al. Anal. Chem. 2008 , 80 , 303 ], the method has been widely deployed for targeted lipid structure elucidation but its application to high throughput and liquid chromatography-based workflows has been limited due to the relatively slow nature of the requisite ion-molecule reactions that result in long ion-trapping times and consequently low instrument duty cycle. Here, the implementation of OzID in a high-pressure region, the ion-mobility spectrometry cell, of a contemporary quadrupole time-of-flight mass spectrometer is described. In this configuration, a high number density of ozone was achieved and thus abundant and diagnostic OzID product ions could be observed even on the timescale of transmission through the reaction region (ca. 20-200 ms), representing a 50-1000-fold improvement in performance over prior OzID implementations. Collisional activation applied prereaction was found to yield complementary and structurally informative product ions arising from ozone- and collision-induced dissociation. Ultimately, the compatibility of this implementation with contemporary ultrahigh performance liquid chromatography is demonstrated with the resulting hyphenated approach showing the ability to separate and uniquely identify isomeric phosphatidylcholines that differ only in their position(s) of unsaturation.

Methyl Transfer in Glucosinolate Biosynthesis Mediated by Indole Glucosinolate O-Methyltransferase 5.

Indole glucosinolates (IGs) are plant secondary metabolites that are derived from the amino acid tryptophan. The product of Arabidopsis (Arabidopsis thaliana) IG core biosynthesis, indol-3-ylmethyl glucosinolate (I3M), can be modified by hydroxylation and subsequent methoxylation of the indole ring in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O-methyltransferases have been identified as key genes for IG modification, our knowledge about the IG modification pathways is not complete. In particular, it is unknown which enzyme is responsible for methyl transfer in the 1-IG modification pathway and whether this pathway plays a role in defense, similar to 4-IG modification. Here, we analyze two Arabidopsis transfer DNA insertion lines with targeted metabolomics. We show that biosynthesis of 1-methoxyindol-3-ylmethyl glucosinolate (1MOI3M) from I3M involves the predicted unstable intermediate 1-hydroxyindol-3-ylmethyl glucosinolate (1OHI3M) and that IGMT5, a gene with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification pathway in Arabidopsis belowground defense.

Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis.

Liver X receptors (Lxrα and Lxrß) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

Sites of metabolic substitution: investigating metabolite structures utilising ion mobility and molecular modelling.

Drug metabolism is an integral part of the drug development and drug discovery process. It is required to validate the toxicity of metabolites in support of safety testing and in particular provide information on the potential to form pharmacologically active or toxic metabolites. The current methodologies of choice for metabolite structural elucidation are liquid chromatography/tandem mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. There are, in certain cases, examples of metabolites whose sites of metabolism cannot be unequivocally identified by MS/MS alone. Utilising commercially available molecular dynamics packages and known quantum chemistry basis sets, an ensemble of lowest energy structures were generated for a group of aromatic hydroxylated metabolites of the model compound ondansetron. Theoretical collision cross-sections were calculated for each structure. Travelling-wave ion mobility (IMS) measurements were also performed on the compounds, thus enabling experimentally derived collision cross-sections to be calculated. A comparison of the theoretical and experimentally derived collision cross-sections were utilised for the accurate assignment of isomeric drug metabolites. The UPLC/IMS-MS method, described herein, demonstrates the ability to measure reproducibly by ion mobility, metabolite structural isomers, which differ in collision cross-section, both theoretical and experimentally derived, by less than 1 Å(2). This application has the potential to supplement and/or complement current methods of metabolite structural characterisation.

Functional characterisation of a SNP in the ABCC11 allele - effects on axillary skin metabolism, odour generation and associated behaviours.

BACKGROUND: A single nucleotide polymorphism (SNP), 538G→A, leading to a G180R substitution in the ABCC11 gene results in reduced concentrations of apocrine derived axillary odour precursors. OBJECTIVE: Determine the axillary odour levels in the SNP ABCC11 genotype variants and to investigate if other parameters associated with odour production are affected. METHODS: Axillary odour was assessed by subjective quantification and gas chromatography headspace analysis. Metabolite profiles, microbiome diversity and personal hygiene habits were also assessed. RESULTS: Axillary odour in the A/A homozygotes was significantly lower compared to the G/A and G/G genotypes. However, the perception-based measures still detected appreciable levels of axillary odour in the A/A subjects. Metabolomic analysis highlighted significant differences in axillary skin metabolites between A/A subjects compared to those carrying the G allele. These differences resulted in A/A subjects lacking specific volatile odourants in the axillary headspace, but all genotypes produced odoriferous short chain fatty acids. Microbiomic analysis revealed differences in the relative abundance of key bacterial genera associated with odour generation between the different genotypes. Deodorant usage indicated a high level of self awareness of axillary odour levels with A/A individuals less likely to adopt personal hygiene habits designed to eradicate/mask its presence. CONCLUSIONS: The SNP in the ABCC11 gene results in lower levels of axillary odour in the A/A homozygotes compared to those carrying the G allele, but A/A subjects still produce noticeable amounts of axillary odour. Differences in axillary skin metabolites, bacterial genera and personal hygiene behaviours also appear to be influenced by this SNP.

Analysis of androgenic steroid Girard P hydrazones using multistage tandem mass spectrometry.

Seven androgenic steroids have been converted into steroid hydrazones using Girard P hydrazine and analysed by electrospray ionisation multistage tandem mass spectrometry. The cationic derivatives 17alpha-testosterone hydrazone, 17beta-nortestosterone hydrazone, 17beta-bolasterone hydrazone, 17alpha-boldenone hydrazone, 17beta-fluoxymesterone hydrazone, 17alpha-trenbolone hydrazone and 4-chloroandrosten-3,17-dione hydrazone show good response in positive ion mode with enhancements for the method of up to 33 times relative to the native species. Detailed characterisation of fragmentation pathways reveals structurally specific ions formed by fragmentation of the hydrazine moiety. Comparison of structural similarities among the androgenic steroids allows recognition of common ions/fragmentation processes as well as analyte-specific transitions. The suitability of the derivatisation approach in the screening of heifer urine for the presence of administered hormones has been demonstrated using partially purified urine spiked with a mixture of androgenic steroids.