Journal of Chromatography B (v.877, #26)
Editorial Board (i).
Lipidomics: Developments and applications by Xianlin Han (2663).
High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry by Marcus Ståhlman; Christer S. Ejsing; Kirill Tarasov; Jeanna Perman; Jan Borén; Kim Ekroos (2664-2672).
Technological advances in mass spectrometry and meticulous method development have produced several shotgun lipidomic approaches capable of characterizing lipid species by direct analysis of total lipid extracts. Shotgun lipidomics by hybrid quadrupole time-of-flight mass spectrometry allows the absolute quantification of hundreds of molecular glycerophospholipid species, glycerolipid species, sphingolipid species and sterol lipids. Future applications in clinical cohort studies demand detailed lipid molecule information and the application of high-throughput lipidomics platforms. In this review we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer. Using this platform to compile comprehensive lipid arrays associated with metabolic dysfunctions is a powerful strategy for pinpointing the mechanistic details by which alterations in tissue-specific lipid metabolism are directly linked to the etiology of many lipid-mediated disorders.
Keywords: Shotgun lipidomics; Automation; High-throughput; Mass spectrometry; Multiple precursor ion scanning; QqTOF; Normal phase HPLC;
Electrospray ionization with low-energy collisionally activated dissociation tandem mass spectrometry of glycerophospholipids: Mechanisms of fragmentation and structural characterization by Fong-Fu Hsu; J. Turk (2673-2695).
This review describes the use of low-energy collisionally activated dissociation (CAD) with both tandem quadrupole and ion-trap mass spectrometry toward structural characterization of glycerophospholipids (GPLs), including classes of glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoserine, glycerophosphoglycerol glycerophosphoinositol and glycerophosphatidic acid, as well as their lyso-, plasmanyl-, and plasmenylphospholipid subclasses. The mechanisms underlying the fragmentation processes leading to structural characterization of GPLs in various ion forms desorbed by electrospray ionization in the positive-ion and negative-ion modes are also discussed. The tandem mass spectrometric approaches afford the identification of the polar head group, the fatty acid substituents and the location of the radyl groups on the glycerol backbone of all the GPLs.
Keywords: Glycerophospholipids; Tandem quadrupole mass spectrometry; Ion-trap mass spectrometry; Electrospray ionization; Charge-remote fragmentation; Charge-driven fragmentation;
Sphingolipidomics: Methods for the comprehensive analysis of sphingolipids by Christopher A. Haynes; Jeremy C. Allegood; Hyejung Park; M. Cameron Sullards (2696-2708).
Sphingolipids comprise a highly diverse and complex class of molecules that serve as both structural components of cellular membranes and signaling molecules capable of eliciting apoptosis, differentiation, chemotaxis, and other responses in mammalian cells. Comprehensive or “sphingolipidomic” analyses (structure specific, quantitative analyses of all sphingolipids, or at least all members of a critical subset) are required in order to elucidate the role(s) of sphingolipids in a given biological context because so many of the sphingolipids in a biological system are inter-converted structurally and metabolically. Despite the experimental challenges posed by the diversity of sphingolipid-regulated cellular responses, the detection and quantitation of multiple sphingolipids in a single sample has been made possible by combining classical analytical separation techniques such as high-performance liquid chromatography (HPLC) with state-of-the-art tandem mass spectrometry (MS/MS) techniques. As part of the Lipid MAPS consortium an internal standard cocktail was developed that comprises the signaling metabolites (i.e. sphingoid bases, sphingoid base-1-phosphates, ceramides, and ceramide-1-phosphates) as well as more complex species such as mono- and di-hexosylceramides and sphingomyelin. Additionally, the number of species that can be analyzed is growing rapidly with the addition of fatty acyl Co-As, sulfatides, and other complex sphingolipids as more internal standards are becoming available. The resulting LC–MS/MS analyses are one of the most analytically rigorous technologies that can provide the necessary sensitivity, structural specificity, and quantitative precision with high-throughput for “sphingolipidomic” analyses in small sample quantities. This review summarizes historical and state-of-the-art analytical techniques used for the identification, structure determination, and quantitation of sphingolipids from free sphingoid bases through more complex sphingolipids such as sphingomyelins, lactosylceramides, and sulfatides including those intermediates currently considered sphingolipid “second messengers”. Also discussed are some emerging techniques and other issues remaining to be resolved for the analysis of the full sphingolipidome.
Keywords: Sphingolipids; Analysis; Mass spectrometry; Tandem mass spectrometry; Liquid chromatography; Electrospray; Nanospray; MALDI;
Phospholipid profiling by tandem mass spectrometry by Zheng Cui; Michael J. Thomas (2709-2715).
This review discusses how to analyze glycerophospholipids from simple lipid extracts without preliminary chromatographic separation. The analytical method emphasized in the review is electrospray ionization with tandem mass spectrometry.
Keywords: Glycerophospholipids; Lipids; Tandem mass spectrometry;
Dynamic lipidomics with stable isotope labelling by Anthony D. Postle; Alan N. Hunt (2716-2721).
Incorporation of stable isotope labelled precursors enables estimation of the kinetics of lipid synthesis and turnover (dynamic lipidomics) in the clinical as well the experimental setting. Recent advances in tandem mass spectrometry extend the analytical possibilities from measurements of isotope enrichments to determinations of intact substrates. Incorporations of deuteriated choline, ethanolamine and inositol can be determined by precursor and neutral loss scans of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, respectively. This experimental approach provides information on the kinetics of individual phospholipid molecular species and has considerable potential to probe diseases of lipid metabolism in vivo.
Keywords: Dynamic lipidomics; Stable isotopes; Lipid metabolism; Phospholipid synthesis;
Identification of double bond position in lipids: From GC to OzID by Todd W. Mitchell; Huong Pham; Michael C. Thomas; Stephen J. Blanksby (2722-2735).
Recent developments in mass spectrometry and chromatography provide new possibilities for the identification and in some instances quantification of a wide range of lipids in complex matrices. These advances in analytical technologies have provided a tantalizing glimpse of the true structural diversity of lipids in nature and have reinvigorated interest in the role of lipids in biology. While technological advances have been impressive, difficulties in the ready identification of sites of unsaturation (i.e., double bond position) within these molecules presents a significant impediment to understanding lipid biochemistry. This is of particular importance given the growing body of literature suggesting that the presence of naturally occurring lipid double bond isomers can have a significant influence, both positive and negative, on the development of pathologies such as cancer, cardiovascular disease and type 2 diabetes. This article provides a critical review of the current suite of analytical approaches to the challenge of identification of the position of carbon–carbon double bonds in intact lipids.
Keywords: ESI–MS; Lipidomics; Lipids; Double bond position;
Targeted quantitative analysis of eicosanoid lipids in biological samples using liquid chromatography–tandem mass spectrometry by Clementina Mesaros; Seon Hwa Lee; Ian A. Blair (2736-2745).
The eicosanoids are a large family of arachidonic acid oxidation products that contain 20 carbon atoms. Cyclooxygenase (COX)-derived eicosanoids have important roles as autacoids involved in the regulation of cardiovascular function and tumor progression. Lipoxygenase (LO)-derived eicosanoids have been implicated as important mediators of inflammation, asthma, cardiovascular disease and cancer. Cytochrome P-450 (P450)-derived eicosanoids are both vasodilators and vasoconstrictors. There is intense interest in the analysis of reactive oxygen species (ROS)-derived isoprostanes (isoPs) because of their utility as biomarkers of oxidative stress. Enzymatic pathways of eicosanoid formation are regioselective and enantioselective, whereas ROS-mediated eicosanoid formation proceeds with no stereoselectivity. Many of the eicosanoids are also present in only pM concentrations in biological fluids. This presents a formidable analytical challenge because methodology is required that can separate enantiomers and diastereomers with high sensitivity and specificity. However, the discovery of atmospheric pressure ionization (API)/MS methodology of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron capture (EC) APCI has revolutionized our ability to analyze endogenous eicosanoids. LC separations of eicosanoids can now be readily coupled with API ionization, collision induced dissociation (CID) and tandem MS (MS/MS). This makes it possible to efficiently conduct targeted eicosanoid analyses using LC-multiple reaction motoring (MRM)/MS. Several examples of targeted eicosanoid lipid analysis using conventional LC-ESI/MS have been discussed and some new data on the analysis of eicosanoids using chiral LC-ECAPCI/MS has been presented.
Keywords: Liquid chromatography–mass spectrometry; Lipids; Fatty acids; Electrospray ionization; Electron capture atmospheric pressure chemical ionization; Hydroxyeicosatetraenoic acids; Isoprostanes; Stable isotopes;
Analysis of endocannabinoids, their congeners and COX-2 metabolites by Philip J. Kingsley; Lawrence J. Marnett (2746-2754).
Since the discovery of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in the early 1990s, the endocannabinoid system has been implicated in a wide array of physiological processes, such as control of food intake and energy balance, fertility and obesity. As the importance of this system becomes apparent, there is a tremendous need for robust, sensitive and efficient analytical methodology for the examination of the endocannabinoids, their congeners and putative metabolites. This review will summarize quantitative analytical methodology as reported in the literature from 1992 to present for the analysis of endocannabinoids and related compounds.
Keywords: Endocannabinoids; Mass spectrometry; 2-Arachidonoyl glycerol; Anandamide; Prostaglandin glycerol; Cyclooxygenase;
Lipidomic analysis of endocannabinoid metabolism in biological samples by Giuseppe Astarita; Daniele Piomelli (2755-2767).
The endocannabinoids are signaling lipids present in many living organisms. They activate G protein-coupled cannabinoid receptors to modulate a broad range of biological processes that include emotion, cognition, inflammation and reproduction. The endocannabinoids are embedded in an interconnected network of cellular lipid pathways, the regulation of which is likely to control the strength and duration of endocannabinoid signals. Therefore, physiopathological or pharmacological perturbations of these pathways may indirectly affect endocannabinoid activity and, vice versa, endocannabinoid activity may influence lipid pathways involved in other metabolic and signaling events. Recent progress in liquid chromatography and mass spectrometry has fueled the development of targeted lipidomic approaches, which allow researchers to examine complex lipid interactions in cells and gain a broader view of the endocannabinoid system. Here, we review these new developments from the perspective of our laboratory's experience in the field.
Keywords: Lipidomics; Anandamide; 2-Arachidonoylglycerol (2-AG); N-acyl phosphatidylethanolamine (NAPE); Fatty-acid amide hydrolase (FAAH); Diacylglycerol (DAG); Monoacylglycerol; Triacylglycerol (TAG); Arachidonic acid; Prostaglandin;
Chromatographic methods for the analyses of 2-halofatty aldehydes and chlorohydrin molecular species of lysophosphatidylcholine by Carolyn J. Albert; Dhanalakshmi S. Anbukumar; Maria C. Messner; David A. Ford (2768-2777).
Plasmalogens are targeted by hypohalous acids resulting in the production of 2-chlorofatty aldehydes, 2-bromofatty aldehydes and chlorohydrin species of lysophosphatidylcholine. These novel lipids may have important roles in the pathophysiological sequelae of cardiovascular diseases as well as serve as biomarkers of cardiovascular disease. Accordingly, the discovery of these new lipid species have required the development of techniques for their purification and quantification. Thin layer chromatography, high performance liquid chromatography (LC) and gas chromatography (GC) of these lipids and their derivatives have provided a battery of tools for their analyses. These lipids have been quantified using flame ionization detection (FID) and mass spectrometry (MS).
Keywords: Plasmalogens; Fatty aldehydes; Chlorohydrins; Myeloperoxidase; Eosinophil peroxidase; Reactive chlorinating species;
Analysis of neurosterols by GC–MS and LC–MS/MS by William J. Griffiths; Yuqin Wang (2778-2805).
The term neurosteroid was coined by Baulieu and colleagues in Paris towards the end of the last century to describe steroids which are synthesised in the central or peripheral nervous system [E.E. Baulieu, Psychoneuroendocrinology 23 (1998) 963–87]. This definition was restricted to side-chain “shortened” steroids with 21 carbon atoms or less, and excluded sterols and their carboxylic acids with an intact side-chain. By analogy, we now use the term neurosterol to describe C27 sterols synthesised in the nervous system. In this review we discuss the biological importance of neurosterols, and how they are extracted, isolated, and analysed by GC–MS and LC–MS/MS, from brain and relevant body fluids. We present applications of methodology employed for analysis of specific sterols and comment on the relative merits of the methods employed. Finally, the importance of future in-depth “sterolomic” investigations of brain is highlighted.
Keywords: Sterol; Mass spectrometry; Lipidomics; Cholesterol; Alzheimer's disease; LXR; Brain; Oxysterol;
Profiling of plant hormones by mass spectrometry by Xiangqing Pan; Xuemin Wang (2806-2813).
Plant hormones regulate various aspects of plant growth and development, and different hormones may interact additively, synergistically, or antagonistically. Mass spectrometry has become a powerful tool for quantitative profiling of multiple classes of plant hormones because of its high sensitivity and selectivity. The capacity to simultaneously quantify multiple classes of phytohormones will facilitate the study of hormone function and cross-talk.
Keywords: ESI-MS/MS; Phytohormones; Arabidopsis thaliana; Plant; Metabolic profiling; Mass spectrometry;
Discovering novel brain lipids by liquid chromatography/tandem mass spectrometry by Ziqiang Guan (2814-2821).
Discovery and structural elucidation of novel brain lipids hold great promise in revealing new lipid functions in the brain and in understanding the biochemical mechanisms underlying brain physiology and pathology. The revived interests in searching for novel brain lipids have been stimulated by the expanding knowledge of the roles of lipids in brain functions, lipids acting as signaling molecules, and the advent of lipidomics enabled by the advances in mass spectrometry (MS) and liquid chromatography (LC). The identification and characterization of two classes of novel lipids from the brain are reviewed here: N-acyl phosphatidylserine (N-acyl-PS) and dolichoic acid (Dol-CA). The identification of these lipids benefited from the use of efficient lipid fractionation and separation techniques and highly sensitive, high-resolution tandem MS.
Keywords: N-Acyl phosphatidylserine; Dolichoic acid; Mass spectrometry; Liquid chromatography;
Direct profiling of tissue lipids by MALDI-TOFMS by Shelley N. Jackson; Amina S. Woods (2822-2829).
Advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have allowed for the direct analysis of biological molecules from tissue. Although most of the early studies of direct tissue profiling by MALDI-TOFMS have focused on proteins and peptides, analysis of lipids has increased dramatically in recent years. This review gives an overview of the factors to consider when analyzing lipids directly from tissue and some recent examples of the use of MALDI-TOFMS for the direct profiling of lipids in tissue.
Keywords: Lipids; Direct profiling; Tissue; MALDI; MS/MS;
Lipid-based biomarkers for cancer by Aaron Zefrin Fernandis; Markus Rene Wenk (2830-2835).
Lipids play important and diverse roles in cells. Most obvious functions are storage of chemical energy, provision of structural support of biological membranes and signaling. All these cellular processes are of critical relevance to cells which undergo transformation, cancer progression and metastasis. Thus, it is likely that certain classes of lipids are reflective for the cellular physiology in cancer cells and tissue. Here we discuss key roles of lipids involved in cancer as well as challenges for development of novel lipid-based biomarkers. Special emphasis will be given to mass spectrometry based analysis of lipids. Such technology has been successfully used for qualitative and quantitative analysis of lipids with very different chemistries. Comparative analysis, often in case–control regimes, and either in non-targeted (e.g. by liquid chromatography–single stage mass spectrometry) or targeted (i.e. by tandem mass spectrometry) fashion yields vast arrays of information. Uni-variate (such as Student's t-test or Mann–Whitney U-test) and multivariate statistics (principal components analysis, machine learning and regression analysis) are next used to identify variations in individual lipid species and/or to lower dimensions for visualization and grouping of cases and controls. As a result surrogate (single or multi-parameter) markers are identified which form the basis for functional validation as well as potential translation to alternative analytical readouts.
Keywords: Lipidomic; Cancer; Mass spectrometry; Multiple reaction monitoring; Biomarker;
Analytical strategies in lipidomics and applications in disease biomarker discovery by Chunxiu Hu; Rob van der Heijden; Mei Wang; Jan van der Greef; Thomas Hankemeier; Guowang Xu (2836-2846).
Lipidomics is a lipid-targeted metabolomics approach aiming at comprehensive analysis of lipids in biological systems. Recently, lipid profiling, or so-called lipidomics research, has captured increased attention due to the well-recognized roles of lipids in numerous human diseases to which lipid-associated disorders contribute, such as diabetes, obesity, atherosclerosis and Alzheimer's disease. Investigating lipid biochemistry using a lipidomics approach will not only provide insights into the specific roles of lipid molecular species in health and disease, but will also assist in identifying potential biomarkers for establishing preventive or therapeutic approaches for human health. Recent technological advancements in mass spectrometry and rapid improvements in chromatographic techniques have led to the rapid expansion of the lipidomics research field. In this review, emphasis is given to the recent advances in lipidomics technologies and their applications in disease biomarker discovery.
Keywords: Lipidomics; Lipid-associated disorders; Biomarkers; Lipid profiling;
Algorithms for automatic processing of data from mass spectrometric analyses of lipids by Haowei Song; Jack Ladenson; John Turk (2847-2854).
Lipidomics comprises large-scale studies of the structures, quantities, and functions of lipid molecular species. Recently developed mass spectrometric methods for lipid analyses, especially electrospray ionization (ESI) tandem mass spectrometry, permit identification and quantitation of an enormous variety of distinct lipid molecular species from small amounts of biological samples but generate a huge amount of experimental data within a brief interval. Processing such data sets so that comprehensible information is derived from them requires bioinformatics tools, and algorithms developed for proteomics and genomics have provided some strategies that can be directly adapted to lipidomics. The structural diversity and complexity of lipids, however, also requires the development and application of new algorithms and software tools that are specifically directed at processing data from lipid analyses. Several such tools are reviewed here, including LipidQA. This program employs searches of a fragment ion database constructed from acquired and theoretical spectra of a wide variety of lipid molecular species, and raw mass spectrometric data can be processed by the program to achieve identification and quantification of many distinct lipids in mixtures. Other approaches that are reviewed here include LIMSA (Lipid Mass Spectrum Analysis), SECD (Spectrum Extraction from Chromatographic Data), MPIS (Multiple Precursor Ion Scanning), FIDS (Fragment Ion Database Searching), LipidInspector, Lipid Profiler, FAAT (Fatty Acid Analysis Tool), and LIPID Arrays. Internet resources for lipid analyses are also summarized.
Keywords: Lipidomics; Electrospray ionization (ESI); Tandem mass spectrometry; Algorithms and software tools;
Bioinformatics and computational methods for lipidomics by Perttu S. Niemelä; Sandra Castillo; Marko Sysi-Aho; Matej Orešič (2855-2862).
Large amounts of lipidomics data are rapidly becoming available. However, there is a lack of tools capable of taking the full advantage of the wealth of new information. Lipid bioinformatics is thus an emerging need as well as challenge for lipid research. Lipid concentration changes in biological systems reflect regulation at multiple spatial and dynamic scales, e.g., biochemical reactions in the cells, intercellular lipid trafficking, changes in cell membrane composition, systemic lipid metabolism or lipid oxidation. In order to address the complexity of lipids and their regulation, four areas of bioinformatics need to be developed: (1) data processing and lipid identification, (2) statistical data analysis, (3) pathway analysis, and (4) lipid modeling in systems and biophysical contexts. In this paper we overview the current state of the lipid bioinformatics field as well as suggest few potential new areas of research.
Keywords: Bioinformatics; Biological membrane; Lipid metabolism; Lipidomics; Liquid chromatography; Mass spectrometry; Metabolic pathways; Metabolomics; Stable isotope tracers; Systems biology;
Mass-spectrometric analysis of hydroperoxy- and hydroxy-derivatives of cardiolipin and phosphatidylserine in cells and tissues induced by pro-apoptotic and pro-inflammatory stimuli by Vladimir A. Tyurin; Yulia Y. Tyurina; Mi-Yeon Jung; Muhammad A. Tungekar; Karla J. Wasserloos; Hülya Bayır; Joel S. Greenberger; Patrick M. Kochanek; Anna A. Shvedova; Bruce Pitt; Valerian E. Kagan (2863-2872).
Oxidation of two anionic phospholipids – cardiolipin (CL) in mitochondria and phosphatidylserine (PS) in extramitochondrial compartments – is important signaling event, particularly during the execution of programmed cell death and clearance of apoptotic cells. Quantitative analysis of CL and PS oxidation products is central to understanding their molecular mechanisms of action. We combined the identification of diverse phospholipid molecular species by ESI-MS with quantitative assessments of lipid hydroperoxides using a fluorescence HPLC-based protocol. We characterized CL and PS oxidation products formed in a model system (cyt c/H2O2), in apoptotic cells (neurons, pulmonary artery endothelial cells) and mouse lung under inflammatory/oxidative stress conditions (hyperoxia, inhalation of single walled carbon nanotubes). Our results demonstrate the usefulness of this approach for quantitative assessments, identification of individual molecular species and structural characterization of anionic phospholipids that are involved in oxidative modification in cells and tissues.
Keywords: Oxidative lipidomics; Cardiolipin; Phosphatidylserine; Phospholipid hydroperoxides; Mass-spectrometry; Cytochrome c; Apoptosis;
A shotgun lipidomics study of a putative lysophosphatidic acid acyl transferase (PlsC) in Sinorhizobium meliloti by Libia Saborido Basconcillo; Rahat Zaheer; Turlough M. Finan; Brian E. McCarry (2873-2882).
A shotgun lipidomics approach was used to study the knockout mutant of a putative lysophosphatidic acyl acid transferase (PlsC) in order to delineate the function of this enzyme in Sinorhizobium meliloti. In plsC knockout mutant lipids that contained 16:0 and 16:1 fatty acids and their biosynthetically related cyclopropane fatty acid (cis-9,10-methylene hexadecanoic acid) decreased up to 93%. Tandem mass spectrometry experiments in the presence of added Li+ showed that the putative PlsC (SMc00714) functioned as a lysophosphatidic acid acyl transferase specific for the transfer of C16 fatty acids to the sn-2 position of lipids. The levels of lipids containing C18 fatty acids were unaffected in plsC mutant, suggesting the presence of one or more fatty acyl transferases in the genome of S. meliloti with selectivity towards C18 fatty acids. Two non-phosphorus containing lipid classes, sulfoquinovosyldiacylglycerol and 1,2-diacylglyceryl-trimethylhomoserine lipids, showed similar decreases in C16 fatty acid content as phospholipids in plsC knockout mutant; these non-phosphorus containing lipids share a common biosynthetic origin with phospholipids, most likely involving phosphatidic acid. Ornithine lipids containing C16 fatty acids also showed decreased levels in PlsC knockout mutant, suggesting that PlsC is also involved in their biosynthesis.
Keywords: Shotgun lipidomics; Phospholipid biosynthesis; Lysophosphatidic acid acyl transferase; Non-phosphorus containing lipids;
Mass spectrometric imaging of lipids using desorption electrospray ionization by Allison L. Dill; Demian R. Ifa; Nicholas E. Manicke; Zheng Ouyang; R. Graham Cooks (2883-2889).
Desorption electrospray ionization (DESI), a relatively new ambient ionization technique used in mass spectrometry (MS), allows for the direct analysis of samples such as thin tissue sections, to be conducted outside of vacuum in the ambient environment and often without sample preparation. DESI-MS has been used in order to systematically characterize phospholipids, which are abundant species in biological tissue samples. Lipids play important biological roles and differences in lipid compositions have been seen in diseases such as cancer and Alzheimer's disease. Imaging of thin tissue sections exploits the ability of DESI-MS to study these lipids directly in the biological matrix. In imaging MS (IMS), a mass spectrum is recorded at each pixel while moving the surface containing the sample so that the entire sample area is covered. The information in these mass spectra can be combined to create a 2D chemical image of the sample, combining information on spatial distribution with information on chemical identity from the characteristic ions in the mass spectra. DESI-MS has been used to image a variety of tissue samples including human liver adenocarcinoma, rat brain, human breast tissue and canine abdominal tumor tissue. Comparisons between diseased and normal tissue are made in these studies.
Keywords: Ambient ionization; Imaging; Lipidomics; Mass spectrometry;
Targeted lipidomics approach for endogenous N-acyl amino acids in rat brain tissue by Bo Tan; Y. William Yu; M. Francesca Monn; H. Velocity Hughes; David K. O’Dell; J. Michael Walker (2890-2894).
Great effort has been devoted to characterize signaling lipids in central nervous system. This has led to a search for novel strategies to characterize hitherto unknown lipid compositions. Here we developed two methods, one for identification and one for quantification, for N-acyl amino acids, a novel lipid family. The identification method contains a series of purification steps followed by nano-LC/MS/MS and high-throughput screening of the datasets with a potent search algorithm based on fragment ion analysis. MS/MS spectra with good quality can be obtained with 150 fmol of targeted lipids on column with our nano-LC/MS/MS. More than one thousand mass spectra generated using the information dependent acquisition mode of Analyst QS software can be analyzed in 1 min using our home built software. The quantification method utilized the multiple reaction monitoring mode in Analyst software to measure the endogenous levels of N-acyl amino acids in rat brain. Using these two methods we were able to identify and quantify 11 previously reported N-acyl amino acids with endogenous levels ranging from 0.26 to 333 pmol g−1 wet rat brain.
Keywords: Targeted lipidomics; N-acyl amino acids; Rat brain;
Specific GC–MS/MS stable-isotope dilution methodology for free 9- and 10-nitro-oleic acid in human plasma challenges previous LC–MS/MS reports by Dimitrios Tsikas; Alexander Zoerner; Anja Mitschke; Yahya Homsi; Frank-Mathias Gutzki; Jens Jordan (2895-2908).
Nitrated unsaturated fatty acids including nitro-oleic acid (NO2-OA) have been measured in human blood samples in their free and esterified forms. Plasma concentrations in healthy subjects have been reported to be of the order of 600 nM for free NO2-OA and 300 nM for esterified NO2-OA, as measured by LC–MS/MS. In the present article we report a GC–MS/MS method for the specific and accurate quantification of two NO2-OA isomers, i.e., 9-NO2-OA and 10-NO2-OA, in human plasma using newly prepared, isolated, characterized and standardized 15N-labeled analogs. This method involves SPE extraction of fatty acids from slightly acidified plasma samples (pH 5), conversion to their pentafluorobenzyl (PFB) esters, isolation by HPLC, solvent extraction from a single HPLC fraction and GC–MS/MS analysis in the electron capture negative-ion chemical ionization (ECNICI) mode. Quantification was performed by selected-reaction monitoring (SRM) of m/z 46 ([NO2]−) and m/z 47 ([15NO2]−) produced by collision-induced dissociation (CID) from the parent ions [M−PFB]− at m/z 326 for endogenous 9-NO2-OA and 10-NO2-OA and m/z 327 for the internal standards 9-15NO2-OA and 10-15NO2-OA. We partially validated the GC–MS/MS method for 9-NO2-OA and 10-NO2-OA in human plasma and quantified these nitro-oleic species in plasma of 15 healthy volunteers. We identified two isomers, i.e., 9-NO2-OA and 10-NO2-OA, in human plasma under physiological conditions and found these nitrated fatty acids at a mean concentration of 1 nM each. This concentration is about 600 times lower than that reported by others using LC–MS/MS. Our GC–MS/MS studies on nitro-oleic acid and 3-nitrotyrosine suggest that the extent of nitration of biomolecules such as unsaturated fatty acids and tyrosine is very low in health. In this article we discuss analytical and biological ramifications potentially associated with measurement of nitrated biomolecules in biological systems.
Keywords: Fatty acids; Lipids; Nitration; Stable isotopes; Standardization; Validation;
Targeted stable-isotope dilution GC–MS/MS analysis of the endocannabinoid anandamide and other fatty acid ethanol amides in human plasma by Alexander A. Zoerner; Frank M. Gutzki; Maria T. Suchy; Bibiana Beckmann; Stefan Engeli; Jens Jordan; Dimitrios Tsikas (2909-2923).
Anandamide (arachidonoyl ethanol amide, AEA) is an endocannabinoid, acting on CB1 and CB2 receptors. Elevated plasma AEA concentrations in humans have been associated amongst others with obesity, psychological disorders and miscarriage. The occurrence in human plasma of ethanol amides of other unsaturated and saturated fatty acids, including oleic acid and palmitic acid, has also been reported. Most data available on anandamide and other fatty acid ethanol amides (FAEA) until now have been generated by using the LC–MS/MS methodology. Here, we describe a stable-isotope dilution GC–MS/MS method for the quantitative determination of AEA, oleic acid ethanol amide (OEA) and palmitic acid ethanol amide (PEA) in human plasma using their stable-isotope labeled analogs as internal standards. Other FAEA were found in plasma and their concentration was estimated. The present method involves a single solvent extraction of FAEA and their internal standards from plasma (50–1000 μl) with toluene, derivatization to the pentafluorobenzamide pentafluoropropionyl derivatives (FAEA–PFBz–PFP), and simultaneous quantification by selected reaction monitoring of the carboxylate anions produced by collision-induced dissociation of the parent ions [M−PFBz]−. The present method was fully validated for anandamide. Thus, accuracy and imprecision of the method were within the range of 100 ± 20% and less than 20%, respectively, in the range investigated (0–4 nM). Mean overall recovery was 90 ± 3%. The LOQ and LOD values of the method were determined to be 0.25 nM of added AEA in plasma samples and 400 amol of injected AEA–PFBz–PFP derivative, respectively. In plasma of 16 healthy individuals AEA concentration was measured to be 1.35 ± 0.32 nM. This finding is concordant to literature AEA plasma concentrations as measured by LC–MS/MS. The plasma concentrations of OEA, PEA and other FAEA are higher than that of AEA. This GC–MS/MS method is straightforward, accurate, precise, highly specific for FAEA and useful in basic and clinical research.
Keywords: Arachidonoyl ethanol amide; AEA; Derivatization; Fatty acid ethanol amide; GC–MS/MS; Validation;
Systematic analysis of choline-containing phospholipids using multi-dimensional mass spectrometry-based shotgun lipidomics by Kui Yang; Zhongdan Zhao; Richard W. Gross; Xianlin Han (2924-2936).
Herein, a systematic study on the identification and quantitation of choline-containing phospholipid molecular species, including choline glycerophospholipid (PC), lysoPC, and sphingomyelin (SM), is described using multi-dimensional mass spectrometry-based shotgun lipidomics after intrasource separation (MDMS-SL). Current methods for analysis of choline-containing lipids were improved through multiple modifications leading to: (1) identification of constituents present in the PC and SM classes, subclasses of PC, and individual molecular species using MDMS-SL analysis in the positive-ion mode; (2) identification of the fatty acyl constituents and their regiospecificity in diacyl PC molecular species through the neutral loss of trimethylamine plus fatty acids; (3) direct identification of the alkenyl chains of plasmenylcholine species using precursor ion scans of the fragment ions carrying the alkenyl chains; (4) elimination of the effects of polyunsaturation on the quantitation of PC species by multiple ratiometric comparisons; (5) accurate identification and quantitation of lysoPC molecular species including regioisomers by diagnostic fragment ions; and (6) accurate identification and quantitation of SM molecular species by neutral loss scans of phosphocholine plus methyl aldehyde which is specific to SM molecular species. With these enhancements, the application of MDMS-SL for the analyses of choline-containing phospholipid molecular species in biomedical research has been extended to a much larger number of molecular species with greater quantitative accuracy and an increased depth of structural information.
Keywords: Choline-containing phospholipid; Lipidomics; Lysoplasmenylcholine; Lysophosphatidylcholine; Multi-dimensional mass spectrometry; Plasmenylcholine; Shotgun lipidomics; Sphingomyelin;