Mass Spectrometry-based Lipidomics Research Articles

Mass Spectrometry-based Lipidomics and Its Application toBiomedical Research

Lipidomics, a branch of metabolomics, is the large-scale study of pathways and networks of all cellular lipids in biological systems such as cells, tissues or organisms. The recent advance in mass spectrometry technologies have enabled more comprehensive lipid profiling in the biological samples. In this review, we compared four representative lipid profiling technoligies including GC-MS, LC-MS, direct infusion-MS and imaging-MS. We also summarized representative lipid database, and further discussed the applications of lipidomics to the diagnostics of various diseases such as diabetes, obesity, hypertension, and Alzheimer diseases.

Lipidomics Profiling and Risk of Cardiovascular Disease in the Prospective Population-Based Bruneck Study

The bulk of cardiovascular disease risk is not explained by traditional risk factors. Recent advances in mass spectrometry allow the identification and quantification of hundreds of lipid species. Molecular lipid profiling by mass spectrometry may improve cardiovascular risk prediction. Lipids were extracted from 685 plasma samples of the prospective population-based Bruneck Study (baseline evaluation in 2000). One hundred thirty-five lipid species from 8 different lipid classes were profiled by shotgun lipidomics with the use of a triple-quadrupole mass spectrometer. Levels of individual species of cholesterol esters (CEs), lysophosphatidylcholines, phosphatidylcholines, phosphatidylethanolamines (PEs), sphingomyelins, and triacylglycerols (TAGs) were associated with cardiovascular disease over a 10-year observation period (2000-2010, 90 incident events). Among the lipid species with the strongest predictive value were TAGs and CEs with a low carbon number and double-bond content, including TAG(54:2) and CE(16:1), as well as PE(36:5) (P=5.1 × 10⁻⁷, 2.2 × 10⁻⁴, and 2.5 × 10⁻³, respectively). Consideration of these 3 lipid species on top of traditional risk factors resulted in improved risk discrimination and classification for cardiovascular disease (cross-validated ΔC index, 0.0210 [95% confidence interval, 0.0010-0.0422]; integrated discrimination improvement, 0.0212 [95% confidence interval, 0.0031-0.0406]; and continuous net reclassification index, 0.398 [95% confidence interval, 0.175-0.619]). A similar shift in the plasma fatty acid composition was associated with cardiovascular disease in the UK Twin Registry (n=1453, 45 cases). This study applied mass spectrometry-based lipidomics profiling to population-based cohorts and identified molecular lipid signatures for cardiovascular disease. Molecular lipid species constitute promising new biomarkers that outperform the conventional biochemical measurements of lipid classes currently used in clinics.

Lipidomic “Deep Profiling”: An Enhanced Workflow to Reveal New Molecular Species of Signaling Lipids

Current mass spectrometry-based lipidomics aims to comprehensively cover wide ranges of lipid classes. We introduce a strategy to capture phospho-monoester lipids and improve the detection of long-chain base phosphates (LCB-Ps, e.g., sphingosine-1-phosphate). Ten novel LCB-Ps (d18:2, t20:1, odd carbon forms) were discovered and characterized in tissues from human and mouse, as well in D. melanogaster and S. cerevisiae. These findings have immediate relevance for our understanding of sphingosine-1-phosphate biosynthesis, signaling, and degradation.

Characterization and Quantification of Diacylglycerol Species in Biological Extracts after One-Step Derivatization: A Shotgun Lipidomics Approach

Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well-known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost-effective and high throughput methodologies for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multidimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/μL), high specificity, and broad linear dynamics range (2500-fold) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) versus a 5-fold difference between 3 month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states.

The Intracellular Bacteria Chlamydia Hijack Peroxisomes and Utilize Their Enzymatic Capacity to Produce Bacteria-Specific Phospholipids

Chlamydia trachomatis is an obligate intracellular pathogen responsible for loss of eyesight through trachoma and for millions of cases annually of sexually transmitted diseases. The bacteria develop within a membrane-bounded inclusion. They lack enzymes for several biosynthetic pathways, including those to make some phospholipids, and exploit their host to compensate. Three-dimensional fluorescence microscopy demonstrates that small organelles of the host, peroxisomes, are translocated into the Chlamydia inclusion and are found adjacent to the bacteria. In cells deficient for peroxisome biogenesis the bacteria are able to multiply and give rise to infectious progeny, demonstrating that peroxisomes are not essential for bacterial development in vitro. Mass spectrometry-based lipidomics reveal the presence in C. trachomatis of plasmalogens, ether phospholipids whose synthesis begins in peroxisomes and have never been described in aerobic bacteria before. Some of the bacterial plasmalogens are novel structures containing bacteria-specific odd-chain fatty acids; they are not made in uninfected cells nor in peroxisome-deficient cells. Their biosynthesis is thus accomplished by the metabolic collaboration of peroxisomes and bacteria.

A role for oleoylethanolamide in chronic lymphocytic leukemia

Oleoylethanolamide (OEA) is a bioactive lipid that stimulates nuclear and G protein-coupled receptors and regulates appetite and fat metabolism. It has not previously been shown to have a role in cancer. However, a mass spectrometry-based lipidomics platform revealed the presence of high amounts of OEA in the plasma of chronic lymphocytic leukemia (CLL) patients compared with normal donors. CLL cells produced OEA and the magnitude of plasma OEA levels was related directly to the circulating leukemic cell number. OEA from CLL cells was increased by URB-597, an inhibitor of fatty acid amide hydrolase (FAAH), and decreased by inflammatory mediators that downregulate expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD). These enzymes degrade and synthesize OEA, respectively. Nonphysiologic doses of OEA prevented spontaneous apoptosis of CLL cells in a receptor-independent manner that was mimicked by its free fatty acid (FFA) derivative oleate. However, OEA-containing supernatants from CLL cells induced lipolysis in adipocytes, lipid products from adipocytes protected CLL cells from cytotoxic chemotherapy, and increased levels of FFAs were found in CLL plasma that correlated with OEA. We suggest OEA is a lipolytic factor produced by CLL cells to fuel their growth with a potential role in drug resistance and cancer cachexia.

Alterations in Mouse Brain Lipidome after Disruption of CST Gene: A Lipidomics Study

To investigate the effects of a critical enzyme, cerebroside sulfotransferase (CST), involving sulfatide biosynthesis on lipid (particularly sphingolipid) homeostasis, herein, we determined the lipidomes of brain cortex and spinal cord from CST null and heterozygous (CST(-/-) and CST(+/-), respectively) mice in comparison to their wild-type littermates by multi-dimensional mass spectrometry-based shotgun lipidomics. As anticipated, we demonstrated the absence of sulfatide in the tissues from CST(-/-) mice and found that significant reduction of sulfatide mass levels was also present, but in an age-dependent manner, in CST(+/-) mice. Unexpectedly, we revealed that the profiles of sulfatide species in CST(+/-) mice were significantly different from that of littermate controls with an increase in the composition of species containing saturated and hydroxylated fatty acyl chains. Contrary to the changes of sulfatide levels, shotgun lipidomics analysis did not detect significant changes of the mass levels of other lipid classes examined. Taken together, shotgun lipidomics analysis demonstrated anticipated sulfatide mass deficiency in CST defect mouse brain and revealed novel brain lipidome homeostasis in these mice. These results might provide new insights into the role of CST in myelin function.

A Role for Peroxisome Proliferator-activated Receptor γ Coactivator 1 (PGC-1) in the Regulation of Cardiac Mitochondrial Phospholipid Biosynthesis

The energy demands of the adult mammalian heart are met largely by ATP generated via oxidation of fatty acids in a high capacity mitochondrial system. Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)-α and -β serve as inducible transcriptional coregulators of genes involved in mitochondrial biogenesis and metabolism. Whether PGC-1 plays a role in the regulation of mitochondrial structure is unknown. In this study, mice with combined deficiency of PGC-1α and PGC-1β (PGC-1αβ(-/-)) in adult heart were analyzed. PGC-1αβ(-/-) hearts exhibited a distinctive mitochondrial cristae-stacking abnormality suggestive of a phospholipid abnormality as has been described in humans with genetic defects in cardiolipin (CL) synthesis (Barth syndrome). A subset of molecular species, containing n-3 polyunsaturated species in the CL, phosphatidylcholine, and phosphatidylethanolamine profiles, was reduced in PGC-1αβ-deficient hearts. Gene expression profiling of PGC-1αβ(-/-) hearts revealed reduced expression of the gene encoding CDP-diacylglycerol synthase 1 (Cds1), an enzyme that catalyzes the proximal step in CL biosynthesis. Cds1 gene promoter-reporter cotransfection experiments and chromatin immunoprecipitation studies demonstrated that PGC-1α coregulates estrogen-related receptors to activate the transcription of the Cds1 gene. We conclude that the PGC-1/estrogen-related receptor axis coordinately regulates metabolic and membrane structural programs relevant to the maintenance of high capacity mitochondrial function in heart.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Plasma lipid profiling in a large population-based cohort

We have performed plasma lipid profiling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 μl of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identified statistically significant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was significantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk.

Abstract 214: Protein Restriction Attenuates Intimal Hyperplasia and Alters Blood Lipid Profiles

Introduction Dietary restriction (DR: reduced enteral intake without malnutrition) diminishes the acute phase response to surgical stress in preclinical models. We hypothesized that short-term preoperative protein-free DR would attenuate the vascular response to injury (intimal hyperplasia: IH) while perturbing systemic circulating lipids. Methods After 2 weeks of 60% fat kcal diet, 8 week old B6D2F1/J mice had ad lib access to a complete 10% fat kcal diet (n=10) or a reduced calorie, protein-free diet (DR group, n=10) 1 week before induction of IH, then an ad lib complete diet. IH was induced via a validated model placing a nylon suture tie around the distal carotid artery and external 35g needle mandrel (outer diameter=0.14mm). Subsequent removal of the mandrel created a focal area of stenosis (~78% lumen diameter/~85% flow reduction). 4 weeks later, tissues were harvested for morphology and immunohistochemistry for CD45. Separately, serum was collected from mice fed a complete or DR diet for 1 week absent any surgical stress (n=20/group) for mass spectrometry-based lipidomics. Results DR mice showed less intimal area (p = 0.032) vs controls with statistically equivalent intimal leukocyte infiltration. DR mice also had significantly larger internal elastic lamina length (p = 0.003), a remodeling measure. DR serum exhibited significant decreases in certain classes of circulating lipids, including a collapse of multiple triglyceride types. Conclusions One week of protein-free DR dramatically decreased circulating lipids and attenuated arterial IH. Preoperative dietary manipulations may offer a practical means of extending durability of vascular interventions.

Simulation of triacylglycerol ion profiles: bioinformatics for interpretation of triacylglycerol biosynthesis

Although the synthesis pathways of intracellular triacylglycerol (TAG) species have been well elucidated, assessment of the contribution of an individual pathway to TAG pools in different mammalian organs, particularly under pathophysiological conditions, is difficult, although not impossible. Herein, we developed and validated a novel bioinformatic approach to assess the differential contributions of the known pathways to TAG pools through simulation of TAG ion profiles determined by shotgun lipidomics. This powerful approach was applied to determine such contributions in mouse heart, liver, and skeletal muscle and to examine the changes of these pathways in mouse liver induced after treatment with a high-fat diet. It was clearly demonstrated that assessment of the altered TAG biosynthesis pathways under pathophysiological conditions can be readily achieved through simulation of lipidomics data. Collectively, this new development should greatly facilitate our understanding of the biochemical mechanisms underpinning TAG accumulation at the states of obesity and lipotoxicity.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

Cardiolipins (CLs) are a family of phospholipids essential for mitochondrial structure and function. The defective remodeling of the CL molecules (i.e., the changing of the fatty acyl [FA] composition) has been linked to human pathology. We examined diet‐mediated CL changes in liver mitochondria from healthy male FBNF1 rats fed 24 different low‐fat, isocaloric diets. Growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry‐based, mitochondrial lipidomics profiling revealed up‐regulation of CLs in rats fed saturated or trans fat‐based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins and decreased ubiquinone Q9, implying a low‐grade mitochondrial redox abnormality and compensatory CL elevation. Extended analysis of the rat liver mitochondrial CL pools has revealed previously unobserved regulation of CL, suggesting that (i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of CL species; (ii) FA incorporation into mature CLs undergoes both positive (~400‐fold) and negative (≥2.4‐fold) regulation; (iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra‐acyl CLs are inversely related, suggesting compensatory regulation.This study was funded by U01‐ES16048 (BSK, PI) and P30‐DK040561 (W. A. Walker, PI).

On the future of mass‐spectrometry‐based lipidomics

Lipids have highly diverse functions that go beyond cellular membrane structure and energy storage. One of the great challenges in lipid research will be to understand how the enormous complexity of lipid homeostasis is maintained. Genetic approaches combined with mass spectrometry-based lipidomics will help to elucidate how cells create and maintain their nonrandom lipid distribution within tissues, cells, organelles and lipid bilayers. Lipid homeostasis is crucial for many cellular processes and we are currently only beginning to understand the specific functions of lipids and the local environment that they create.

Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism.

Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.

Global Phospholipidomics Analysis Reveals Selective Pulmonary Peroxidation Profiles upon Inhalation of Single-Walled Carbon Nanotubes

It is commonly believed that nanomaterials cause nonspecific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in the two most abundant phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This nonrandom peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H(2)O(2)/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria-targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung.

Multi‐dimensional mass spectrometry‐based shotgun lipidomics and novel strategies for lipidomic analyses

Since our last comprehensive review on multi-dimensional mass spectrometry-based shotgun lipidomics (Mass Spectrom. Rev. 24 (2005), 367), many new developments in the field of lipidomics have occurred. These developments include new strategies and refinements for shotgun lipidomic approaches that use direct infusion, including novel fragmentation strategies, identification of multiple new informative dimensions for mass spectrometric interrogation, and the development of new bioinformatic approaches for enhanced identification and quantitation of the individual molecular constituents that comprise each cell's lipidome. Concurrently, advances in liquid chromatography-based platforms and novel strategies for quantitative matrix-assisted laser desorption/ionization mass spectrometry for lipidomic analyses have been developed. Through the synergistic use of this repertoire of new mass spectrometric approaches, the power and scope of lipidomics has been greatly expanded to accelerate progress toward the comprehensive understanding of the pleiotropic roles of lipids in biological systems.

Metabolomics in early Alzheimer's disease: identification of altered plasma sphingolipidome using shotgun lipidomics.

BackgroundThe development of plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in Alzheimer's disease (AD). Alterations in ceramides and sphingomyelins have been postulated to play a role in amyloidogensis and inflammatory stress related neuronal apoptosis; however few studies have conducted a comprehensive analysis of the sphingolipidome in AD plasma using analytical platforms with accuracy, sensitivity and reproducibility.Methods and FindingsWe prospectively analyzed plasma from 26 AD patients (mean MMSE 21) and 26 cognitively normal controls in a non-targeted approach using multi-dimensional mass spectrometry-based shotgun lipidomics [1], [2] to determine the levels of over 800 molecular species of lipids. These data were then correlated with diagnosis, apolipoprotein E4 genotype and cognitive performance. Plasma levels of species of sphingolipids were significantly altered in AD. Of the 33 sphingomyelin species tested, 8 molecular species, particularly those containing long aliphatic chains such as 22 and 24 carbon atoms, were significantly lower (p<0.05) in AD compared to controls. Levels of 2 ceramide species (N16:0 and N21:0) were significantly higher in AD (p<0.05) with a similar, but weaker, trend for 5 other species. Ratios of ceramide to sphingomyelin species containing identical fatty acyl chains differed significantly between AD patients and controls. MMSE scores were correlated with altered mass levels of both N20:2 SM and OH-N25:0 ceramides (p<0.004) though lipid abnormalities were observed in mild and moderate AD. Within AD subjects, there were also genotype specific differences.ConclusionsIn this prospective study, we used a sensitive multimodality platform to identify and characterize an essentially uniform but opposite pattern of disruption in sphingomyelin and ceramide mass levels in AD plasma. Given the role of brain sphingolipids in neuronal function, our findings provide new insights into the AD sphingolipidome and the potential use of metabolomic signatures as peripheral biomarkers.

Eosinophils promote resolution of acute peritonitis by producing proresolving mediators in mice

Acute inflammation in healthy individuals is self-limiting and has an active termination program. The mechanisms by which acute inflammation is resolved are of interest. In murine zymosan-induced peritonitis, we found that eosinophils are recruited to the inflamed loci during the resolution phase of acute inflammation. In vivo depletion of eosinophils caused a resolution deficit, namely impaired lymphatic drainage with reduced appearance of phagocytes carrying engulfed zymosan in the draining lymph node, and sustained numbers of polymorphonuclear leukocytes in inflamed tissues. Liquid chromatography-tandem mass spectrometry-based lipidomics of the resolving exudates revealed that locally activated eosinophils in the resolution phase produced proresolving mediators, including protectin D1 (PD1) from docosahexaenoic acid. The resolution deficit caused by eosinophil depletion was rescued by eosinophil restoration or the administration of PD1. Eosinophils deficient in 12/15-lipoxygenase could not rescue the resolution phenotype. The present results indicate that mouse eosinophils and eosinophil-derived lipid mediators, including PD1, have a role in promoting the resolution of acute inflammation, expanding the roles of eosinophils in host defense and resolution.