Search

Highly unsaturated fatty acids (HUFAs) are vulnerable to oxygen attack, thus making HUFA-rich, high metabolic rate/reactive oxygen species (ROS)-generating neurological tissue particularly susceptible to increased oxidative stress. Lipid oxidation is a putative early stage marker of neurodegenerative diseases, suggesting that reliable monitoring of oxidized neural lipids in vivo reveals early pathogenesis. Here, we present a novel methodology to detect and quantify intact in vivo ROS-driven peroxidized phospholipids (LPOx-PLs) in bovine retina extract. A protocol for preparing autoxidized pure phospholipids (PLs) and complex retinal extracts served as reference standards and was adapted to enable analytical parameter development. Fatty acid profiles of bovine retinas were first established with routine gas chromatography (GC) methods and used to customize mass spectrometry scanning for major HUFA-carrying PLs in the retinal extract. Targeted multiple reaction monitoring (MRM) scanning via triple quadrupole tandem mass spectrometry detected native (unoxidized) and oxidation-damaged PL regardless of the position of the O or O 2 addition along the acyl chains and enabled quantification of relative signals from intact native and oxidized PL (5%-10% CV). MRM-triggered information-dependent acquisition (IDA) spectra confirmed the structure of peroxidized PLs, revealing that peroxidized species (+O-OH) dominated over single O-added species in vitro and in vivo . Positive identification and relative quantification are reported for 12 selected in vivo native and peroxidized phosphatidylcholines and phosphatidylethanolamines. These results enable future studies of the initial peroxidation due to toxins, genetics, or other initiating events influencing in vivo oxidation levels and potentially the effectiveness of strategies to mitigate this mechanism of action.

We developed a structural identification method for eicosanoids with various ring structures using mass spectrometry. We discovered that an electron beam with a kinetic energy of 10 eV, which is in the Electron Impact Excitation of Ions from Organics (EIEIO) regime, cleaved the fatty acids enough to distinguish constitutional and cis/trans isomers. In addition to EIEIO, a comparison to authentic standards using differential mobility spectrometry (DMS) can identify diastereomers, which was difficult by EIEIO. The combination of EIEIO and DMS can provide a high-throughput method to identify complete structures of eicosanoids in mixed samples, which is not allowed with conventional analytical methods though eicosanoids are important signaling molecules in biosystems.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: WJG and YW are listed as inventors on the patent “Kit and method for quantitative detection of steroids” US9851368B2. WJG, EY and YW are shareholders in CholesteniX Ltd., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Heavy alcohol drinking dysregulates lipid metabolism, promoting hepatic steatosis - the first stage of alcohol-related liver disease (ALD). The molecular circadian clock plays a major role in synchronizing daily rhythms in behavior and metabolism and clock disruption can cause pathology, including liver disease. Previous studies indicate that alcohol consumption alters liver clock function, but the impact alcohol or clock disruption, or both have on the temporal control of hepatic lipid metabolism and injury remains unclear. Here, we undertook studies to determine whether genetic disruption of the liver clock exacerbates alterations in lipid metabolism and worsens steatosis in alcohol-fed mice. To address this question, male liver-specific Bmal1 knockout (LKO) and flox/flox (Fl/Fl) control mice were fed a control or alcohol-containing diet for 5 weeks. Alcohol significantly dampened diurnal rhythms of mRNA levels in clock genes Bmal1 and Dbp , phase advanced Nr1d1 /REV-ERBα, and induced arrhythmicity in Clock , Noct , and Nfil3 /E4BP4, with further disruption in livers of LKO mice. Alcohol-fed LKO mice exhibited higher plasma triglyceride (TG) and different time-of-day patterns of hepatic TG and macrosteatosis, with elevated levels of small droplet macrosteatosis compared to alcohol-fed Fl/Fl mice. Diurnal rhythms in mRNA levels of lipid metabolism transcription factors ( Srebf1 , Nr1h2 , and Ppara ) were significantly altered by alcohol and clock disruption. Alcohol and/or clock disruption significantly altered diurnal rhythms in mRNA levels of fatty acid (FA) synthesis and oxidation ( Acaca/b , Mlycd , Cpt1a , Fasn , Elovl5/6 , and Fads1/2 ), TG turnover ( Gpat1 , Agpat1/2 , Lpin1/2 , Dgat2 , and Pnpla2/3 ), and lipid droplet ( Plin2/5 , Lipe , Mgll , and Abdh5 ) genes, along with protein abundances of p-ACC, MCD, and FASN. Lipidomics analyses showed that alcohol, clock disruption, or both significantly altered FA saturation and remodeled the FA composition of the hepatic TG pool, with higher percentages of several long and very long chain FA in livers of alcohol-fed LKO mice. In conclusion, these results show that the liver clock is important for maintaining temporal control of hepatic lipid metabolism and that disrupting the liver clock exacerbates alcohol-related hepatic steatosis., (Copyright © 2020 Valcin, Udoh, Swain, Andringa, Patel, Al Diffalha, Baker, Gamble and Bailey.)

Lipidomic analyses aim for absolute quantification of lipid species profiles in biological samples. In past years, mass spectrometry (MS) methods based on high resolution accurate masses (HRAM) have increasingly been applied to identify and quantify lipid species on the MS level. This strategy requires consideration of isobaric overlaps which may also result from various adduct ions. Generally applied solvent additives favor the formation of protonated and ammoniated ions in positive ion mode, yet sodiated ions are also frequently observed. These sodiated ions interfere with protonated ions of the species of the same lipid class with two additional CH 2 and three double bonds (Δ m / z = 0.0025) and the first isotopic peak overlaps with ammoniated ions of a species with one additional CH 2 and four double bonds (Δ m / z = 0.0057). In this work, we present an algorithm based on the sodiated to protonated/ammoniated adduct ion ratios of applied internal standards to correct for these interferences. We could demonstrate that these ratios differ significantly between lipid classes but are affected by neither chain length nor number of double bonds within a lipid class. Finally, the algorithm is demonstrated for correcting human serum samples analyzed by Fourier-transform mass spectrometry (FTMS). Here, the application of sodium correction significantly reduced overestimations and misidentifications.

Modification of the host lipidome via secreted enzymes is an integral, but often overlooked aspect of bacterial pathogenesis. In the current era of prevalent antibiotic resistance, knowledge regarding critical host pathogen lipid interactions has the potential for use in developing novel antibacterial agents. While most studies to date on this matter have focused on specific lipids, or select lipid classes, this provides an incomplete picture. Modern methods of untargeted lipidomics have the capacity to overcome these gaps in knowledge and provide a comprehensive understanding of the role of lipid metabolism in the pathogenesis of infections. In an attempt to determine the role of lipid modifying enzymes produced by staphylococci, we exposed bovine heart lipids, a standardized model for the mammalian lipidome, to spent medium from staphylococcal cultures, and analyzed lipid molecular changes by MS/MSALL shotgun lipidomics. We elucidate distinct effects of different staphylococcal isolates, including 4 clinical isolates of the pathogenic species Staphylococcus aureus, a clinical isolate of the normally commensal species S. epidermidis, and the non-pathogenic species S. carnosus. Two highly virulent strains of S. aureus had a more profound effect on mammalian lipids and modified more lipid classes than the other staphylococcal strains. Our studies demonstrate the utility of the applied untargeted lipidomics methodology to profile lipid changes induced by different bacterial secretomes. Finally, we demonstrate the promise of this lipidomics approach in assessing the specificity of bacterial enzymes for mammalian lipid classes. Our data suggests that there may be a correlation between the bacterial expression of lipid-modifying enzymes and virulence, and could facilitate the guided discovery of lipid pathways required for bacterial infections caused by S. aureus and thereby provide insights into the generation of novel antibacterial agents., Competing Interests: PRSB was employed by SCIEX LLC. during the course of the study. SCIEX allowed PRSB to generate early proof of concept data using their in house instrument platform. PRSB is currently an employee at Avanti Polar Lipids. Lipidomics Consulting Ltd is solely owned and operated by KE. There are no patents, products in development or marketed products to declare. The commercial affiliations of the authors PRSB and KE does not alter our adherence to all the PLOS ONE policies on sharing data and materials. None of the other authors have any competing interests. All the data has been made available with this manuscript.

We report a method for comprehensive structural characterization of lipids in animal tissues using a combination of differential ion mobility spectrometry (DMS) with electron-impact excitation of ions from organics (EIEIO) mass spectrometry. Singly charged lipid ions in protonated or sodiated forms were dissociated by an electron beam having a kinetic energy of 10 eV in a branched radio-frequency ion trap. We established a comprehensive set of diagnostics to characterize the structures of glycerophospholipids, sphingolipids, and acylglycerols, including glycosylated, plasmalogen, and ester forms. This EIEIO mass spectrometer was combined with DMS as a separation tool to analyze complex lipid extracts. Deuterated quantitative standards, which were added during extraction, allowed for the quantitative analysis of the lipid molecular species in various lipid classes. We applied this technique to the total lipids extracted from porcine brain, and we structurally characterized over 300 lipids (with the exception of cis / trans double-bond isomerism in the acyl chains). The structural dataset of the lipidomes, whose regioisomers were distinguished, exhibit a uniquely defined distribution of acyl chains within each lipid class; that is, sn -1 and sn -2 in the cases of glycerophospholipids or sn -2 and ( sn -1, sn -3) in the cases of triacylglycerols., (Copyright © 2018 Baba et al.)

We present a mass spectrometry-based method for the identification of cis and trans double-bond isomers within intact complex lipid mixtures using electron impact excitation of ions from organics (EIEIO) mass spectrometry. EIEIO involves irradiating singly charged lipid ions with electrons having kinetic energies of 5-16 eV. The resulting EIEIO spectra can be used to discern cis and trans double-bond isomers by virtue of the differences in the fragmentation patterns at the carbon-carbon single bonds neighboring the double bonds. For trans double bonds, these characteristic fragments include unique closed-shell and open-shell (radical) products. To explain this fragmentation pattern in trans double bonds, we have proposed a reaction mechanism involving excitation of the double bond's π electrons followed by hydrogen atom rearrangement. Several lipid standards were analyzed using the EIEIO method, including mixtures of these standards. Prior to EIEIO, some of the lipid species in these mixtures were separated from their isomeric forms by using differential mobility spectrometry (DMS). For example, mixed cis and trans forms of triacylglycerols and phosphatidylcholines were identified by this DMS-EIEIO workflow. With this combined gas-phase separation and subsequent fragmentation, we could eliminate the need for authentic standards for identification. When DMS could not separate cis and trans isomers completely, as was the case with sphingomyelins, we relied upon the aforementioned diagnostic EIEIO fragment peaks to determine the relative contribution of the trans double-bond isomer in the mixed samples. We also applied the DMS-EIEIO methodology to natural samples extracted from a ruminant (bovine), which serve as common origins of trans fatty acids in a typical Western diet that includes dairy products.

Collision-induced dissociation (CID) is the most common tool for molecular analysis in mass spectrometry to date. However, there are difficulties associated with many applications because CID does not provide sufficient information to permit details of the molecular structures to be elucidated, including post-translational-modifications in proteomics, as well as isomer differentiation in metabolomics and lipidomics. To face these challenges, we are developing fast electron-based dissociation devices using a novel radio-frequency ion trap ( i.e. , a branched ion trap). These devices have the ability to perform electron capture dissociation (ECD) on multiply protonated peptide/proteins; in addition, the electron impact excitation of ions from organics (EIEIO) can be also performed on singly charged molecules using such a device. In this article, we review the development of this technology, in particular on how reaction speed for EIEIO analyses on singly charged ions can be improved. We also overview some unique, recently reported applications in both lipidomics and glycoproteomics.