Strategies to Improve/Eliminate the Limitations in Shotgun Lipidomics

Direct infusion‐based shotgun lipidomics is one of the most powerful and useful tools in comprehensive analysis of lipid species from lipid extracts of various biological samples with high accuracy/precision. However, despite many advantages, the classical shotgun lipidomics suffers some general dogmas of limitations, such as ion suppression, ambiguous identification of isobaric/isomeric lipid species, and ion source–generated artifacts, restraining the applications in analysis of low‐abundance lipid species, particularly those less ionizable or isomers that yield almost identical fragmentation patterns. This article reviews the strategies (such as modifier addition, prefractionation, chemical derivatization, charge feature utilization) that have been employed to improve/eliminate these limitations in modern shotgun lipidomics approaches (e.g., high mass resolution mass spectrometry–based and multidimensional mass spectrometry–based shotgun lipidomics). Therefore, with the enhancement of these strategies for shotgun lipidomics, comprehensive analysis of lipid species including isomeric/isobaric species is achieved in a more accurate and effective manner, greatly substantiating the aberrant lipid metabolism, signaling trafficking, and homeostasis under pathological conditions.     

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.     

Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair

Ceramides (CERs) play key roles in signal transduction and cell regulation, probably during the keratinization of human hair. Current methods using mass spectrometry (MS), however, are not sufficient to allow the comprehensive analysis of CER molecules, including isobaric and isomeric CERs. Therefore, a method for the comprehensive profiling of CERs was developed. The method developed is based on reversed-phase liquid chromatography (RPLC) coupled to atmospheric pressure chemical ionization (APCI)-MS. Comprehensive identification and profiling of CERs is achieved using two sets of multimass chromatograms obtained from two channel detections that monitor both molecular-related and sphingoid-related ions under two different in-source collision-induced dissociation conditions and using retention times obtained from RPLC. The application of this method revealed that human hair contains 73 species of CER molecules, which were all corroborated by structural analysis using tandem mass spectrometry. The results further revealed that the composition is characterized by predominant molecules consisting of even carbon atom-containing saturated/unsaturated nonhydroxy or alpha-hydroxy fatty acids and C(18) dihydrosphingosine, a minor but distinct content of isobaric/isomeric and odd chain-containing CERs. This successfully developed RPLC-APCI-MS technique allows the comprehensive profiling of CER molecules in hair for the investigation of their physicochemical and physiological roles.     

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.     

Mass spectrometry imaging and profiling of single cells

Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies-secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)-are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enables new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.     

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.     

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.     

Rapid characterization of the fatty acyl composition of complex lipids by collision-induced dissociation time-of-flight mass spectrometry

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for approximately 1 min per sample.     

Advances in chemical proteomic evaluation of lipid kinases—DAG kinases as a case study

Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry–based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase–focused investigations.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

An enhanced plant lipidomics method based on multiplexed liquid chromatography–mass spectrometry reveals additional insights into cold‐ and drought‐induced membrane remodeling

Within the lipidome of plants a few bulk molecular species hamper the detection of the rest, which are present at relatively low levels. In addition, low‐abundance species are often masked by numerous isobaric interferences, such as those caused by isoelemental species and isotopologues. This scenario not only means that minor species are underrepresented, but also leads to potential misidentifications and limits the structural information gathered by lipidomics approaches. In order to overcome these limitations we have developed a multiplexed liquid chromatography–mass spectrometry lipidomics platform able to achieve an enhanced coverage of plant lipidomes. The platform is based on a single extraction step followed by a series of ultra‐performance liquid chromatography separations. Post‐column flow is then directed to both a triple quadrupole analyzer for targeted profiling and a time‐of‐flight analyzer for accurate mass analysis. As a proof of concept, plants were subjected to cold or drought, which are known to trigger widespread remodeling events in plant cell membranes. Analysis of the leaf lipidome yielded 393 molecular species within 23 different lipid classes. This enhanced coverage allowed us to identify lipid molecular species and even classes that are altered upon stress, allowing hypotheses on role of glycosylinositolphosphoceramides (GIPC), steryl glycosides (SG) and acylated steryl glycosides (ASG) in drought stress to be addressed and confirming the findings from numerous previous studies with a single, wide‐ranging lipidomics approach. This extended our knowledge on membrane remodeling during the drought response, integrating sphingolipids and sterol lipids into the current glycerolipid‐based model.     

脂质组学在医药研究中的应用

脂质组学是对整体脂质进行系统分析的一门新兴学科,通过比较不同生理状态下脂代谢网络的变化,进而识别代谢调控中关键的脂生物标志物,最终揭示脂质在各种生命活动中的作用机制。电喷雾电离-质谱技术是脂质组学领域中最核心的研究手段,目前已能对各种脂质尤其是磷脂进行高分辨率、高灵敏度、高通量的分析。随着质谱技术的进步,脂质组学在疾病脂生物标志物的识别、疾病诊断、药物靶点及先导化合物的发现和药物作用机制的研究等方面已展现出广泛的应用前景。     

Spatial mapping of lipids at cellular resolution in embryos of cotton

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.     

Applications of thin layer chromatography,high performance liquid chromatography and mass spectrometry in the fermentation and isolation of the antibiotic nybomycin

Summary Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.     

Profiling lipid changes in plant response to low temperatures

Changes in membrane lipid composition play multiple roles in plant adaptation and survival in the face of chilling and freezing damage. An electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based approach has been used to quantitatively profile membrane lipid molecular species in plant response to low temperatures. This method involves the direct infusion of unfractionated lipid extracts into a mass spectrometer in the precursor and neutral loss scanning modes to identify and quantify lipid species. The profiling analysis reveals significant and distinct lipid changes during cold acclimation and freezing. Comparative profiling of wildtype and mutants provides information about the metabolic and cellular functions of specific phospholipase D genes and enzymes.     

Shotgun lipidomics on high resolution mass spectrometers

Despite their compositional complexity, lipidomes comprise a large number of isobaric species that cannot be distinguished by conventional low resolution mass spectrometry and therefore in-depth MS/MS analysis was required for their accurate quantification. Here we argue that the progress in high resolution mass spectrometry is changing the concept of lipidome characterization. Because exact masses of isobaric species belonging to different lipid classes are not necessarily identical, they can now be distinguished and directly quantified in total lipid extracts. By streamlining and simplifying the molecular characterization of lipidomes, high resolution mass spectrometry has developed into a generic tool for cell biology and molecular medicine.     

A matter of fat: an introduction to lipidomic profiling methods

In recent years, lipidomics or lipid profiling, an extension of metabolomics where the lipid complement of a cell, tissue or organism is measured, has been the recipient of increasing attention as a research tool in a range of diverse disciplines including physiology, lipid biochemistry, clinical biomarker discovery and pathology. The advancement of the field has been driven by the development of analytical technologies, and in particular advances in liquid chromatography mass spectrometry and chemometric methods. In this review, we give an overview of the current methods with which lipid profiling is being performed. The benefits and shortcomings of mass spectrometry both in the presence and absence of chromatographic separation techniques such as liquid-, gas- and thin layer chromatography are explored. Alone these techniques have their limitations but through a combination many of the disadvantages may be overcome providing a valuable analytical tool for a variety of disease processes.     

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.     

Analysis of cholesteryl esters and diacylglycerols using lithiated adducts and electrospray ionization-tandem mass spectrometry

Cholesteryl ester (CE) and diacylglycerol (DAG) molecular species are important lipid storage and signaling molecules. Mass spectrometric analyses of these lipids are complicated by the presence of isobaric molecular ions shared by these lipid classes and by relatively poor electrospray ionization, which is a consequence of an inherently weak dipole moment in these lipid classes. The current study demonstrates that lithiated adducts of CE and DAG molecular ions have enhanced ionization and lipid class-specific fragmentation in tandem mass spectrometry (MS/MS) scan modes, thereby allowing the implementation of strategies capable of lipid class-specific detection. Using neutral loss (NL) mode for the loss of cholestane from cholesterol esters (NL 368.5) and specific selected reaction monitoring for DAG molecular species, the response of specific molecular species to that of internal standards was determined. CE and DAG molecular species were quantified in human coronary artery endothelial cells (HCAECs) incubated with both palmitic acid and oleic acid. Furthermore, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species. Taken together, these studies demonstrate a new analytical approach to assessing CE and DAG molecular species that exploits the utility of lithiated adducts in conjunction with MS/MS approaches.