Three-dimensional visualization of membrane phospholipid distributions in Arabidopsis thaliana seeds: A spatial perspective of molecular heterogeneity

Arabidopsis thaliana has been widely used as a model plant to study acyl lipid metabolism. Seeds of A. thaliana are quite small (approximately 500 × 300 μm and weigh ~ 20 μg), making lipid compositional analyses of single seeds difficult to achieve. Here we have used matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to map and visualize the three-dimensional spatial distributions of two common membrane phospholipid classes, phosphatidylcholine (PC) and phosphatidylinositol (PI), in single A. thaliana seeds. The 3D images revealed distinct differences in distribution of several molecular species of both phospholipids among different seed tissues. Using data from these 3D reconstructions, the PC and PI mol% lipid profiles were calculated for the embryonic axis, cotyledons, and peripheral endosperm, and these data agreed well with overall quantification of these lipids in bulk seed extracts analyzed by conventional electrospray ionization-mass spectrometry (ESI-MS). In addition, MALDI-MSI was used to profile PC and PI molecular species in seeds of wild type, fad2–1, fad3–2, fad6–1, and fae1–1 acyl lipid mutants. The resulting distributions revealed previously unobserved changes in spatial distribution of several lipid molecular species, and were used to suggest new insights into biochemical heterogeneity of seed lipid metabolism. These studies highlight the value of mass spectrometry imaging to provide unprecedented spatial and chemical resolution of metabolites directly in samples even as small as a single A. thaliana seeds, and allow for expanded imaging of plant metabolites to improve our understanding of plant lipid metabolism from a spatial perspective.     

Imaging lipids in biological samples with surface-assisted laser desorption/ionization mass spectrometry: A concise review of the last decade

Knowing the spatial location of the lipid species present in biological samples is of paramount importance for the elucidation of pathological and physiological processes. In this context, mass spectrometry imaging (MSI) has emerged as a powerful technology allowing the visualization of the spatial distributions of biomolecules, including lipids, in complex biological samples. Among the different ionization methods available, the emerging surface-assisted laser desorption/ionization (SALDI) MSI offers unique capabilities for the study of lipids. This review describes the specific advantages of SALDI-MSI for lipid analysis, including the ability to perform analyses in both ionization modes with the same nanosubstrate, the detection of lipids characterized by low ionization efficiency in MALDI-MS, and the possibilities of surface modification to improve the detection of lipids. The complementarity of SALDI and MALDI-MSI is also discussed. Finally, this review presents data processing strategies applied in SALDI-MSI of lipids, as well as examples of applications of SALDI-MSI in biomedical lipidomics.     

Lipidomics in tissues, cells and subcellular compartments

Mass spectrometry (MS) advances in recent years have revolutionized the biochemical analysis of lipids in plants, and made possible new theories about the structural diversity and functional complexity of lipids in plant cells. Approaches have been developed to profile the lipidome of plants with increasing chemical and spatial resolution. Here we highlight a variety of methods for lipidomics analysis at the tissue, cellular and subcellular levels. These procedures allow the simultaneous identification and quantification of hundreds of lipids species in tissue extracts by direct-infusion MS, localization of lipids in tissues and cells by laser desorption/ionization MS, and even profiling of lipids in individual subcellular compartments by direct-organelle MS. Applications of these approaches to achieve improved understanding of plant lipid metabolism, compartmentation and function are discussed.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Mapping of phospholipids by MALDI imaging (MALDI-MSI): realities and expectations

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a novel powerful MS methodology that has the ability to generate both molecular and spatial information within a tissue section. Application of this technology as a new type of biochemical lipid microscopy may lead to new discoveries of the lipid metabolism and biomarkers associated with area-specific alterations or damage under stress/disease conditions such as traumatic brain injury or acute lung injury, among others. However there are limitations in the range of what it can detect as compared with liquid chromatography-MS (LC-MS) of a lipid extract from a tissue section. The goal of the current work was to critically consider remarkable new opportunities along with the limitations and approaches for further improvements of MALDI-MSI. Based on our experimental data and assessments, improvements of the spectral and spatial resolution, sensitivity and specificity towards low abundance species of lipids are proposed. This is followed by a review of the current literature, including methodologies that other laboratories have used to overcome these challenges.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

基于电喷雾电离串联质谱技术的植物脂质组学研究进展

研究表明,脂质不但参与植物的信号转导、小泡运输、细胞骨架重组等多种细胞过程,而且在植物的生长发育和胁迫反应中具有重要作用．但是脂质本身的多样性、复杂性、以及分析手段的滞后限制了人们对脂质的深入认识．电喷雾电离串联质谱(ESI-MS/MS)技术作为一种直接进样的高通量分析技术,能够在短时间内对大多数脂质的不同分子种进行定量分析,极大地方便了人们了解植物因环境变化和生长发育引起的组织内脂质分子种的微量变化．近年来,该技术在植物上的成功应用,推动植物脂质组学研究取得了重要进展,揭示出脂质在植物的逆境胁迫反应、防御反应中的多种功能,促进了植物脂质代谢相关基因的鉴定．而且,该技术与其他脂质分析技术结合,促使人们在脂质的分布、运输、转化和新脂质种类的鉴定方面有新的进展．概要介绍了ESI-MS/MS技术的特点,重点综述了该技术在植物脂质组学研究中的应用进展,并展望了该技术今后的发展方向.     

Deep-lipidotyping by mass spectrometry: recent technical advances and applications

In-depth structural characterization of lipids is an essential component of lipidomics. There has been a rapid expansion of mass spectrometry methods that are capable of resolving lipid isomers at various structural levels over the past decade. These developments finally make deep-lipidotyping possible, which provides new means to study lipid metabolism and discover new lipid biomarkers. In this review, we discuss recent advancements in tandem mass spectrometry (MS/MS) methods for identification of complex lipids beyond the species (known headgroup information) and molecular species (known chain composition) levels. These include identification at the levels of carbon-carbon double bond (C=C) location and sn-position, as well as characterization of acyl chain modifications. We also discuss the integration of isomer-resolving MS/MS methods with different lipid analysis workflows and their applications in lipidomics. The results showcase the distinct capabilities of deep-lipidotyping in untangling the metabolism of individual isomers and sensitive phenotyping by using relative fractional quantitation of the isomers.     

Applications of nuclear magnetic resonance in lipid analyses: An emerging powerful tool for lipidomics studies

The role of lipids in cell, tissue, and organ physiology is crucial; as many diseases, including cancer, diabetes, neurodegenerative, and infectious diseases, are closely related to absorption and metabolism of lipids. Mass spectrometry (MS) based methods are the most developed powerful tools to study the synthetic pathways and metabolic networks of cellular lipids in biological systems; leading to the birth of an emerging subject lipidomics, which has been extensively reviewed. Nuclear magnetic resonance (NMR), another powerful analytical tool, which allows the visualization of single atoms and molecules, is receiving increasing attention in lipidomics analyses. However, very little work focusing on lipidomic studies using NMR has been critically reviewed. This paper presents a first comprehensive summary of application of ¹H, ¹³C & ³¹P NMR in lipids and lipidomics analyses. The scientific basis, principles and characteristic diagnostic peaks assigned to specific atoms/molecular structures of lipids are presented. Applications of 2D NMR in mapping and monitoring of the components and their changes in complex lipids systems, as well as alteration of lipid profiling over disease development are also reviewed. The applications of NMR lipidomics in diseases diagnosis and food adulteration are exemplified.     

Integration of lipidomics and metabolomics for in-depth understanding of cellular mechanism and disease progression

Mass spectrometry(MS)-based omics technologies are now widely used to profile small molecules in multiple matrices to confer comprehensive snapshots of cellular metabolic phenotypes.The metabolomes of cells,tissues,and organisms comprise a variety of molecules including lipids,amino acids,sugars,organic acids,and so on.Metabolomics mainly focus on the hydrophilic classes,while lipidomics has emerged as an independent omics owing to the complexities of the organismal lipidomes.The potential roles of lipids and small metabolites in disease pathogenesis have been widely investigated in various human diseases,but system-level understanding is largely lacking,which could be partly attributed to the insufficiency in terms of metabolite coverage and quantitation accuracy in current analytical technologies.While scientists are continuously striving to develop high-coverage omics approaches,integration of metabolomics and lipidomics is becoming an emerging approach to mechanistic investigation.Integration of metabolome and lipidome offers a complete atlas of the metabolic landscape,enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology,facilitating the study of interconnection between lipids and other metabolites in disease progression.In this review,we summarize omics-based findings on the roles of lipids and metabolites in the pathogenesis of selected major diseases threatening public health.We also discuss the advantages of integrating lipidomics and metabolomics for in-depth understanding of molecular mechanism in disease pathogenesis.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics

By using shotgun lipidomics based on the separation of lipid classes in the electrospray ion source (intrasource separation) and two-dimensional (2D) MS techniques (Han, X., and R. W. Gross. 2004. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. First published on June 18, 2004; doi: 10.1002/mas.20023, In press), individual molecular species of most major and many minor lipid classes can be quantitated directly from biological lipid extracts. Herein, we extended shotgun lipidomics to the characterization and quantitation of cerebroside molecular species in biological samples. By exploiting the differential fragmentation patterns of chlorine adducts using electrospray ionization (ESI) tandem mass spectrometry, hydroxy and nonhydroxy cerebroside species are readily identified. The hexose (either galactose or glucose) moiety of a cerebroside species can be distinguished by examination of the peak intensity ratio of its product ions at m/z 179 and 89 (i.e., 0.74 +/- 0.10 and 4.8 +/- 0.7 for galactose- and glucose-containing cerebroside species, respectively). Quantitation of cerebroside molecular species (as little as 10 fmol) from chloroform extracts of brain tissue samples was directly conducted by 2D ESI/MS after correction for differences in (13)C-isotopomer intensities. This method was demonstrated to have a greater than 1,000-fold linear dynamic range in the low concentration region; therefore, it should have a wide range of applications in studies of the cellular sphingolipid lipidome.     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.     

New approach for glyco- and lipidomics--molecular scanning of human brain gangliosides by TLC-Blot and MALDI-QIT-TOF MS

We have developed a TLC-Blot system that makes possible the direct analysis of blotted glycosphingolipids on a polyvinylidene difluoride membrane from a high-performance TLC plate by immunological staining, chemical staining, enzymatic treatment and mass spectrometric (MS) analysis. An ion trap type matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) MS apparatus improved not only the molecular identification but also the analysis of molecular species of lipids on the polyvinylidene difluoride membrane. A new approach for glyco- and lipidomics, molecular scanning technology by a combination of TLC-Blot and MALDI-QIT-TOF MS, was developed and applied to human brain gangliosides separated from the tissues of patients with neural diseases and control patients. The results clearly showed a change of ganglioside composition, in addition to identifying individual ganglioside molecular species, in the hippocampus gray matter of patients with Alzheimer's disease. The results strongly suggested that metabolic changes of gangliosides played an important role in the progression of this disease. The present technology with molecular imaging should provide valuable information for elucidating the significance of molecular species in neuronal functions such as neural transmission, memory, and learning.     

Lipid Atlas of Keratinocytes and Betulin Effects on its Lipidome Profiled by Comprehensive UHPLC–MS/MS with Data Independent Acquisition Using Targeted Data Processing

Betulin is a pentacyclic triterpene with demonstrated healing properties in mid‐dermal wounds. A few earlier studies have provided insights into the wound healing effects on the molecular level. However, there are still questions left on the molecular targets of betulin. Therefore, a pharmacolipidomics analysis of betulin is undertaken in human immortalized keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. For this purpose, lipid extracts of keratinocytes treated with betulin and untreated controls are comprehensively analyzed by an untargeted UHPLC–ESI–QTOF‐MS/MS lipidomics profiling workflow using data‐independent acquisition. Targeted data processing allows the identification of 611 lipid species from 21 different lipid classes. Statistical analysis of the identified lipids shows significant changes in 440 lipid species that can be described as downregulation of cholesteryl esters and triacylglycerides and upregulation of glycerophospholipids, sphingolipids, and diacylglycerides. Additionally, some other signals corresponding to triterpenes are found in the betulin group and suggested that betulin is incorporated (in the membrane) and metabolized in keratinocytes.     

Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles

Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed‐specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. Here we show by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of the oilseed crop Camelina sativa, indicating tissue‐specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over‐expression of an acyl–acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in Camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.     

Visualization of lipid droplet composition by direct organelle mass spectrometry

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

Expanding the horizons of lipidomics. Towards fluxolipidomics

Abstract

This short review takes into consideration the status of lipidomics as issued from almost a decade of development. Because of the huge number of molecular species analyzed, there is a trend in subdividing lipidomics according to subdomains, in particular relating to the function of molecules. It is also pointed out that lipid imaging without the use of exogenous probes will help making relationships between molecular structures and the topography of lipid assemblies, especially in cellular compartments. Finally, a fluxomics approach is proposed for lipid molecular species, both in terms of compartments and biochemical metabolism. The example of fluxolipidomics of essential fatty acids toward their enzyme-dependent oxygenated metabolites and further toward their degradation products is developed.