Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes

Top–down shotgun lipidomics relies on direct infusion of total lipid extracts into a high‐resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top–down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high‐resolution spectra in both polarity modes with sub‐ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle. Copyright © 2012 John Wiley & Sons, Ltd.     

Software tool for mining liquid chromatography/multi‐stage mass spectrometry data for comprehensive glycerophospholipid profiling

Electrospray ionization mass spectrometry (ESI‐MS) has emerged as an indispensable tool in the field of lipidomics. Despite the growing interest in lipid analysis, there are only a few software tools available for data evaluation, as compared for example to proteomics applications. This makes comprehensive lipid analysis a complex challenge. Thus, a computational tool for harnessing the raw data from liquid chromatography/mass spectrometry (LC/MS) experiments was developed in this study and is available from the authors on request. The Profiler‐Merger‐Viewer tool is a software package for automatic processing of raw‐data from data‐dependent experiments, measured by high‐performance liquid chromatography hyphenated to electrospray ionization hybrid linear ion trap Fourier transform mass spectrometry (FTICR‐MS and Orbitrap) in single and multi‐stage mode. The software contains three parts: processing of the raw data by Profiler for lipid identification, summarizing of replicate measurements by Merger and visualization of all relevant data (chromatograms as well as mass spectra) for validation of the results by Viewer. The tool is easily accessible, since it is implemented in Java and uses Microsoft Excel (XLS) as output format. The motivation was to develop a tool which supports and accelerates the manual data evaluation (identification and relative quantification) significantly but does not make a complete data analysis within a black‐box system. The software's mode of operation, usage and options will be demonstrated on the basis of a lipid extract of baker's yeast (S. cerevisiae). In this study, we focused on three important representatives of lipids: glycerophospholipids, lyso‐glycerophospholipids and free fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Profiling of lipids in Leishmania donovani using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry

There is evidence from our current research on resistance to stibigluconate and from some previous observations that lipid composition may be altered in resistant Leishmania donovani and in order to explore this we required a comprehensive lipidomics method. Phospholipids can be analysed by direct infusion into a mass spectrometer and such methods can work very well. However, chromatographic methods can also be very effective and are extensively used. They potentially avoid ion suppression effects, associate lipid classes with a retention time range and deliver good quantitative accuracy. In the current study three chromatography columns were compared for their ability to separate different classes of lipid. Butylsilane (C‐4), Zic‐HILIC and a silica gel column were compared. The best results were obtained with a silica gel column used in hydrophilic interaction chromatography (HILIC) mode with a mobile phase gradient consisting of (A) 20% isopropyl alcohol (IPA) in acetonitrile (v/v) and (B) 20% IPA in 0.02 M ammonium formate. Using these conditions separate peaks were obtained for triglycerides (TG), phosphoinositols (PI), inositol phosphoceramides (IPC), phosphatidylethanolamines (PE), phosphatidylserines (PS), phosphatidylcholines (PC), sphingosines (SG), lysophosphatidyethanolamines (LPE) and lysophosphatidylcholines (LPC). The methodology was applied to the analysis of lipid extracts from Leishmania donovani and by coupling the chromatography with an LTQ Orbitrap mass spectrometer. It was possible to detect 188 lipid species in the extracts with the following breakdown: PC 59, PE 38, TG 35, PI 20, CPI 13, LPC 11, LPE 2 and SG 10. The fatty acid composition of the more abundant lipids was characterised by MS² and MS³ experiments carried out by using an LCQ Deca low‐resolution ion trap instrument coupled with the silica gel column. The separation of lipids into well‐defined groups gives extra confidence in their identification and minimises the risk of ion suppression effects. High‐resolution mass spectrometry was necessary in order to be able to differentiate between acyl‐ and acyl‐alkyl‐lipids. Copyright © 2010 John Wiley & Sons, Ltd.     

Multidimensional mass spectrometry-based shotgun lipidomics analysis of vinyl ether diglycerides

Diglycerides play a central role in lipid metabolism and signaling in mammalian cells. Although diacylglycerol molecular species comprise the majority of cellular diglycerides that are commonly measured using a variety of approaches, identification of extremely low abundance vinyl ether diglycerides has remained challenging. In this work, representative molecular species from the three diglyceride subclasses (diacyl, vinyl ether, and alkyl ether diglycerides; hereafter referred to as diradylglycerols) were interrogated by mass spectrometric analysis. Product ion mass spectra of the synthesized diradylglycerols with varied chain lengths and degrees of unsaturation demonstrated diagnostic fragmentation patterns indicative of each subclass. Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) analysis of mouse brain and heart lipid extracts were performed using the identified informative signature product ions. Through an array of tandem mass spectrometric analyses utilizing the orthogonal characteristics of neutral loss scanning and precursor ion scanning, the differential fragmentation of each subclass was exploited for high-yield structural analyses. Although molecular ion mass spectra readily identified diacylglycerol molecular species directly from the hexane fractions of tissue extracts enriched in nonpolar lipids, molecular ion peaks corresponding to ether-linked diglycerides were not observable. The power of MDMS-SL utilizing the tandem mass spectrometric array analysis was demonstrated by identification and profiling of individual molecular species of vinyl ether diglycerides in mouse brain and heart from their undetectable molecular ion peaks during MS¹ analysis. Collectively, this technology enabled the identification and profiling of previously inaccessible vinyl ether diglyceride molecular species in mammalian tissues directly from extracts of biologic tissues.

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Paternò-Büchi(PB)反应与串联质谱结合实现不饱和脂质精确结构解析

脂质在一系列关键生命过程中发挥着重要作用。结构决定功能,因此脂质结构解析与功能研究一直受到研究者的关注。尽管质谱已成为脂质分析的最常用技术之一,但脂质的精细结构解析仍面临诸多挑战。为解决上述问题,将Paternò-Büchi(PB)光化学反应与串联质谱相结合,实现了不饱和脂质中碳碳双键■的定位以及不饱和脂质■位置异构体的定量分析。此外,该技术可与鸟枪法、液相色谱-质谱和质谱成像等技术联用,用于复杂生物样品中脂质的分析。     

1H-Pteridine-2,4-dione (lumazine): a new MALDI matrix for complex (phospho)lipid mixtures analysis

Nowadays, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry represents an emerging and versatile tool for analysis of lipids. However, direct (i.e., with no previous separation of lipid classes) analysis of crude extracts containing a complex mixture of lipids (a problem typically encountered in shotgun lipidomics) is still a quite challenging task using a conventional MALDI matrix such as 2,5-dihydroxybenzoic acid (DHB). Indeed, in the presence of phospholipids containing quaternary ammonium groups, such as phosphatidylcholines and sphingomyelins, strong ionization-suppression effects are experienced especially in positive ion mode. To overcome this limitation, lumazine (1H-pteridine-2,4-dione) was evaluated as an alternative matrix. Lumazine in the solid state showed an absorption maximum at 350 nm, ionizes/desorbs without appreciable decomposition and extensive cluster formation, and can be used in both ion modes. In positive ion mode, the main species were M ^•+ and 2M ^•+ radical cations and cationized species ([M+H]⁺, [M+Na]⁺, [M+2Na+2Li-3H]⁺). In negative ion mode, the main signals observed were the deprotonated molecular ion and the radical anion. The signal-to-noise ratio for phosphatidylglycerols and phosphatidylethanolamines using lumazine was almost 1 order of magnitude higher than that observed for DHB. Lumazine was successfully used for MALDI analysis (positive and negative ion modes) of crude lipid extracts of milk, soymilk, and hen egg, where phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols could additionally be detected.     

Recent progresses of derivatization approaches in the targeted lipidomics analysis by mass spectrometry

Lipidomics plays an essential role in the development of an improved understanding of lipids metabolism and the identification of new biomarkers or therapeutic targets of related diseases. The strong analytical power of mass spectrometry and its rapid developments in the respect of instruments and techniques have significantly accelerated the emerging lipidomics and related application fields in biology, medicine, and pharmacy. The strategy of chemical derivatization can remarkably improve the shortcomings of mass spectrometric analytical technologies of shotgun lipidomics and liquid chromatography mass spectrometry, and in the past decade many related studies have been reported for fatty acids, glycerophospholipids, sphingomyelins, monoglycerides, diacylglycerols, long‐chain bases, steroids, and so on. Therefore, this review will focus on new chemical derivatization approaches about the research progresses of shotgun‐based and liquid chromatography mass spectrometry–based targeted lipidomics (from 2005 to July 2019, most of reports emerged in the past 5 years), and put forward the problems and prospects in this field. It is expected to be helpful for the design and synthesis of new derivatization reagents, especially the outstanding stable isotope labeling derivatization reagents, and the development and application of new chemical derivatization strategies and matched mass spectrometric analysis methods.     

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) in combination with high‐performance thin‐layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and ‐sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post‐source decay (PSD) product ion analysis was performed in order to obtain further structural information (head‐ and acyl‐group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R‐250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI‐MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on‐line HPTLC/MALDI‐MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI‐MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 µL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF‐MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

The cell wall of the pathogenic bacterium Streptococcus pneumoniae contains glucopyranosyl diacylglycerol (GlcDAG) and galactoglucopyranosyldiacylglycerol (GalGlcDAG). The specific GlcDAG consisting of vaccenic acid substituent at sn‐2 was recently identified as another glycolipid antigen family recognized by invariant natural killer T‐cells. Here, we describe a linear ion‐trap multiple‐stage (MSⁿ) mass spectrometric approach towards structural analysis of GalGlcDAG and GlcDAG. Structural information derived from MSⁿ (n = 2, 3) on the [M + Li]⁺ adduct ions desorbed by electrospray ionization affords identification of the fatty acid substituents, assignment of the fatty acyl groups on the glycerol backbone, as well as the location of double bond along the fatty acyl chain. The identification of the fatty acyl groups and determination of their regio‐specificity were confirmed by MSⁿ (n = 2, 3) on the [M + NH₄]⁺ ions. We establish the structures of GalGlcDAG and GlcDAG isolated from S. pneumoniae, in which the major species consists of a 16:1‐ or 18:1‐fatty acid substituent mainly at sn‐2, and the double bond of the fatty acid is located at ω‐7 (n‐7). More than one isomers were found for each mass in the family. This mass spectrometric approach provides a simple method to achieve structure identification of this important lipid family that would be very difficult to define using the traditional method. Copyright © 2012 John Wiley & Sons, Ltd.     

Mono‐, di‐ and trimethylated homologues of isoprenoid tetraether lipid cores in archaea and environmental samples: mass spectrometric identification and significance

Higher homologues of widely reported C₈₆ isoprenoid diglycerol tetraether lipid cores, containing 0–6 cyclopentyl rings, have been identified in (hyper)thermophilic archaea, representing up to 21% of total tetraether lipids in the cells. Liquid chromatography‐tandem mass spectrometry confirms that the additional carbon atoms in the C_87‐88 homologues are located in the etherified chains. Structures identified include dialkyl and monoalkyl (‘H‐shaped’) tetraethers containing C_40‐42 or C_81‐82 hydrocarbons, respectively, many representing novel compounds. Gas chromatography‐mass spectrometric analysis of hydrocarbons released from the lipid cores by ether cleavage suggests that the C₄₀ chains are biphytanes and the C₄₁ chains 13‐methylbiphytanes. Multiple isomers, having different chain combinations, were recognised among the dialkyl lipids. Methylated tetraethers are produced by Methanothermobacter thermautotrophicus in varying proportions depending on growth conditions, suggesting that methylation may be an adaptive mechanism to regulate cellular function. The detection of methylated lipids in Pyrobaculum sp. AQ1.S2 and Sulfolobus acidocaldarius represents the first reported occurrences in Crenarchaeota. Soils and aquatic sediments from geographically distinct mesotemperate environments that were screened for homologues contained monomethylated tetraethers, with di‐ and trimethylated structures being detected occasionally. The structural diversity and range of occurrences of the C_87‐89 tetraethers highlight their potential as complementary biomarkers for archaea in natural environments. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry-based lipidomics reveals key lipid molecules as potential therapeutic targets of Polygonum cuspidatum against hyperlipidemia in a hamster model

Polygonum cuspidatum is a homology of traditional medicine and functional food widely distributed around the world. Our previous study on the hyperlipidemic animal model demonstrated that Polygonum cuspidatum was effective in ameliorating hyperlipidemia, which is characterized by lipid disorders. Herein, the regulatory effect of Polygonum cuspidatum on lipid metabolism needs to be known if its hypolipidemic mechanism is desired to clarify. In this study, an ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry-based lipidomic strategy was first applied to investigate the lipidomic patterns of high-fat diet-induced hyperlipidemic hamsters when treated with Polygonum cuspidatum. The results showed that Polygonum cuspidatum improved the lipidomic profile of hyperlipidemia. A total of 65 differential lipids related to the hypolipidemic effect of Polygonum cuspidatum were screened out and identified, and these differential lipids covered various categories, such as phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, sphingomyelins and so on. Orally administrated Polygonum cuspidatum restored these differential lipids back to normal or nearly normal levels. This study adopted lipidomics to reveal the key lipid molecules as potential therapeutic targets of Polygonum cuspidatum against hyperlipidemia, which would provide a scientific basis for its clinical application.     

Development of an in vitro system combining aqueous and lipid phases as a tool to understand gastric nitrosation

Nitrite has long been considered a potential pre‐carcinogen for gastric cancer. Acidification of salivary nitrite, derived from dietary nitrate, produces nitrosative species such as NOSCN, NO⁺ and N₂O₃, which can form potentially carcinogenic N‐nitroso compounds. Ascorbic acid inhibits nitrosation by converting the nitrosative species into nitric oxide (NO). However, NO diffuses rapidly to adjacent lipids, where it reacts with oxygen to reform nitrosative species. Nitrosation has been studied in vitro in aqueous systems and less frequently in organic systems; however, there is a need to investigate acid‐catalysed nitrosation in a system combining aqueous and lipid environments, hence providing a physiologically relevant model. Here, we describe a two‐phase system, which can be used as a tool to understand acid‐catalysed nitrosation. Using gas chromatography/ion trap tandem mass spectrometry, we investigated the nitrosation of secondary amines as a function of the lipid phase composition and reaction mixing. An increased interface surface area was a driver for nitrosation, while incorporation of unsaturated fatty acids affected morpholine and piperidine nitrosation differently. Linoleic acid methyl esters did not affect morpholine nitrosation and only had a limited effect on N‐nitrosopiperidine formation, while incorporation of free linoleic acid to the lipid phase significantly reduced N‐nitrosopiperidine formation, but increased N‐nitrosomorpholine formation at low levels. The mechanisms driving these effects are thought to involve amine partitioning, polarity and unsaturated fatty acids acting as scavengers of nitrosating species, findings relevant to the nitrosative chemistry occurring in the stomach, where the gastric acid meets a range of dietary fats which are emulsified during digestion. Copyright © 2010 John Wiley & Sons, Ltd.     

How does a CC double bond cleave in the gas phase? Fragmentation of protonated ketotifen in mass spectrometry

In the literature, it is reported that the protonated ketotifen mainly undergoes C?C double bond cleavage in electrospray ionization tandem mass spectrometry (ESI‐MS/MS); however, there is no explanation on the mechanism of this fragmentation reaction. Therefore, we carried out a combined experimental and theoretical study on this interesting fragmentation reaction. The fragmentation of protonated ketotifen (m/z 310) always generated a dominant fragment ion at m/z 96 in different electrospray ionization mass spectrometers (ion trap, triple quadrupole and linear trap quadrupole (LTQ)‐orbitrap). The mechanism of the generation of this product ion (m/z 96) through the C?C double bond cleavage was proposed to be a sequential hydrogen migration process (including proton transfer, continuous two‐step 1,2‐hydride transfer and ion‐neutral complex‐mediated hydride transfer). This mechanism was supported by density functional theory (DFT) calculations and a deuterium labeling experiment. DFT calculations also showed that the formation of the product ion m/z 96 was most favorable in terms of energy. This study provides a reasonable explanation for the fragmentation of protonated ketotifen in ESI‐MS/MS, and the fragmentation mechanism is suitable to explain other C?C double bond cleavage reactions in mass spectrometry. Copyright © 2016 John Wiley & Sons, Ltd.     

表面修饰探针纳升电喷雾质谱脂质组学对大型溞和蚤状溞的快速鉴别

采用表面修饰探针敞开式纳升电喷雾质谱技术建立了脂质组学方法,以实现溞属(Daphnia)生物大型溞和蚤状溞的快速鉴别.采用微尺度表面修饰探针直接从Daphnia体内萃取和富集出脂质化合物,然后在常压敞开条件下进行纳升电喷雾质谱分析,利用高分辨率质谱仪获得脂质指纹图谱并进行脂质种类和结构鉴定.采用该方法获得了大型溞和蚤状溞的脂质指纹图谱,并研究了大型溞和蚤状溞体内的脂质组成特征.通过结合化学计量学手段实现了大型溞和蚤状溞的快速区分,并鉴定了相关的生物标志物.结果表明,所建立的微萃取探针纳升电喷雾质谱脂质组学方法具有分析速度快、灵敏度高的优点,可用于Daphnia属生物的快速鉴定和识别.     

Rapid profiling of polymeric phenolic acids in Salvia miltiorrhiza by hybrid data‐dependent/targeted multistage mass spectrometry acquisition based on expected compounds prediction and fragment ion searching

Phenolic acids are the major water‐soluble components in Salvia miltiorrhiza (>5%). According to previous studies, many of them contribute to the cardiovascular effects and antioxidant effects of S. miltiorrhiza. Polymeric phenolic acids can be considered as the tanshinol derived metabolites, e.g., dimmers, trimers, and tetramers. A strategy combined with tanshinol‐based expected compounds prediction, total ion chromatogram filtering, fragment ion searching, and parent list‐based multistage mass spectrometry acquisition by linear trap quadropole‐orbitrap Velos mass spectrometry was proposed to rapid profile polymeric phenolic acids in S. miltiorrhiza. More than 480 potential polymeric phenolic acids could be screened out by this strategy. Based on the fragment information obtained by parent list‐activated data dependent multistage mass spectrometry acquisition, 190 polymeric phenolic acids were characterized by comparing their mass information with literature data, and 18 of them were firstly detected from S. miltiorrhiza. Seven potential compounds were tentatively characterized as new polymeric phenolic acids from S. miltiorrhiza. This strategy facilitates identification of polymeric phenolic acids in complex matrix with both selectivity and sensitivity, which could be expanded for rapid discovery and identification of compounds from complex matrix.     

Mass spectrometry based lipid(ome) analyzer and molecular platform: a new software to interpret and analyze electrospray and/or matrix‐assisted laser desorption/ionization mass spectrometric data of lipids: a case study from Mycobacterium tuberculosis

Mass Spectrometry based Lipid(ome) Analyzer and Molecular Platform (MS‐LAMP) is a new software capable of aiding in interpreting electrospray ionization (ESI) and/or matrix‐assisted laser desorption/ionization (MALDI) mass spectrometric data of lipids. The graphical user interface (GUI) of this standalone programme is built using Perl::Tk. Two databases have been developed and constituted within MS‐LAMP, on the basis of Mycobacterium tuberculosis (M. tb) lipid database ( www.mrl.colostate.edu ) and that of Lipid Metabolites and Pathways Strategy Consortium (LIPID MAPS; www.lipidmaps.org ). Different types of queries entered through GUI would interrogate with a chosen database. The queries can be molecular mass(es) or mass‐to‐charge (m/z) value(s) and molecular formula. L IPID M APS identifier also can be used to search but not for M. tb lipids. Multiple choices have been provided to select diverse ion types and lipids. Satisfying to input parameters, a glimpse of various lipid categories and their population distribution can be viewed in the output. Additionally, molecular structures of lipids in the output can be seen using ChemSketch ( www.acdlabs.com ), which has been linked to the programme. Furthermore, a version of MS‐LAMP for use in Linux operating system is separately available, wherein PyMOL can be used to view molecular structures that result as output from General Lipidome MS‐LAMP. The utility of this software is demonstrated using ESI mass spectrometric data of lipid extracts of M. tb grown under two different pH (5.5 and 7.0) conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Multiple‐stage linear ion‐trap with high resolution mass spectrometry towards complete structural characterization of phosphatidylethanolamines containing cyclopropane fatty acyl chain in Leishmania infantum

The structures of phosphatidylethanolamine (PE) in Leishmania infantum are unique in that they consist of a rare cyclopropane fatty acid (CFA) containing PE subfamily, including CFA‐containing plasmalogen PE species. In this contribution, we applied multiple‐stage linear ion‐trap combined with high‐resolution mass spectrometry to define the structures of PEs that were desorbed as [M – H]^? and [M – H + 2Li]⁺ ions by ESI, respectively. The structural information arising from MSⁿ on both the molecular species are complimentary, permitting complete determination of PE structures, including the identities of the fatty acid substituents and their location on the glycerol backbone, more importantly, the positions of the double bond(s) and of the cyclopropane chain of the fatty acid chain, directing to the realization of the CFA biosynthesis pathways that were reported previously. We also uncovered the presence of a minor dimethyl‐PE subclass that has not been previously reported in L. infantum. This LIT MSⁿ mass spectrometric approach led to unambiguous identification of PE molecules including many isomers in complex mixture that would otherwise be very difficult to define using other analytical approaches. Copyright © 2014 John Wiley & Sons, Ltd.     

Enhanced Lipidome Coverage in Shotgun Analyses by using Gas-Phase Fractionation

A high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging.

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Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MSⁿ by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.     

Effect of Headgroups on Small‐Ion Permeability across Archaea‐Inspired Tetraether Lipid Membranes

This paper examines the effects of four different polar headgroups on small‐ion membrane permeability from liposomes comprised of Archaea‐inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane‐leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer‐forming 1‐palmitoyl‐2‐oleoyl‐sn‐glycerol (PO) lipids varied by as much as 32‐fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.