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.     

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.     

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.     

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.     

Is the clinical lipidomics a potential goldmine?

Clinical lipidomics is a new extension of lipidomics to study lipid profiles, pathways, and networks by characterizing and quantifying the complete lipid molecules in cells, biopsy, or body fluids of patients. It undoubtfully has more values if lipidomics can be integrated with the data of clinical proteomic, genomic, and phenomic profiles. A number of challenges, e.g., instability, specificity, and sensitivity, in lipidomics have to be faced and overcome before clinical application. The association of lipidomics data with gene expression and sequencing of lipid-specific proteins/enzymes should be furthermore clarified. Therefore, clinical lipidomics is expected to be more stable during handling, sensitive in response to changes, specific for diseases, efficient in data analyses, and standardized in measurements, in order to meet clinical needs. Clinical lipidomics will become a more important approach in clinical applications and will be the part of “natural” measures for early diagnosis and progress of disease. Thus, clinical lipidomics will be one of the most powerful approaches for disease-specific diagnosis and therapy, once the mystery of lipidomic profiles and metabolic enzymes is deciphered.     

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.     

Novel advances in shotgun lipidomics for biology and medicine

The field of lipidomics, as coined in 2003, has made profound advances and been rapidly expanded. The mass spectrometry-based strategies of this analytical methodology-oriented research discipline for lipid analysis are largely fallen into three categories: direct infusion-based shotgun lipidomics, liquid chromatography-mass spectrometry-based platforms, and matrix-assisted laser desorption/ionization mass spectrometry-based approaches (particularly in imagining lipid distribution in tissues or cells). This review focuses on shotgun lipidomics. After briefly introducing its fundamentals, the major materials of this article cover its recent advances. These include the novel methods of lipid extraction, novel shotgun lipidomics strategies for identification and quantification of previously hardly accessible lipid classes and molecular species including isomers, and novel tools for processing and interpretation of lipidomics data. Representative applications of advanced shotgun lipidomics for biological and biomedical research are also presented in this review. We believe that with these novel advances in shotgun lipidomics, this approach for lipid analysis should become more comprehensive and high throughput, thereby greatly accelerating the lipidomics field to substantiate the aberrant lipid metabolism, signaling, trafficking, and homeostasis under pathological conditions and their underpinning biochemical mechanisms.     

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.     

The foundations and development of lipidomics

For over a century, the importance of lipid metabolism in biology was recognized but difficult to mechanistically understand due to the lack of sensitive and robust technologies for identification and quantification of lipid molecular species. The enabling technological breakthroughs emerged in the 1980s with the development of soft ionization methods (Electrospray Ionization and Matrix Assisted Laser Desorption/Ionization) that could identify and quantify intact individual lipid molecular species. These soft ionization technologies laid the foundations for what was to be later named the field of lipidomics. Further innovative advances in multistage fragmentation, dramatic improvements in resolution and mass accuracy, and multiplexed sample analysis fueled the early growth of lipidomics through the early 1990s. The field exponentially grew through the use of a variety of strategic approaches, which included direct infusion, chromatographic separation, and charge-switch derivatization, which facilitated access to the low abundance species of the lipidome. In this Thematic Review, we provide a broad perspective of the foundations, enabling advances, and predicted future directions of growth of the lipidomics field.     

NIST lipidomics workflow questionnaire: an assessment of community-wide methodologies and perspectives

Introduction

Efforts to harmonize lipidomic methodologies have been limited within the community. Here, we aimed to capitalize on the recent National Institute of Standards and Technology lipidomics interlaboratory comparison exercise by implementing a questionnaire that assessed current methodologies, quantitation strategies, standard operating procedures (SOPs), and quality control activities employed by the lipidomics community.

Objectives

Lipidomics is a rapidly developing field with diverse applications. At present, there are no community-vetted methods to assess measurement comparability or data quality. Thus, a major impetus of this questionnaire was to profile current efforts, highlight areas of need, and establish future objectives in an effort to harmonize lipidomics workflows.

Methods

The 54-question survey inquired about laboratory demographics, lipidomic methodologies and SOPs, analytical platforms, quantitation, reference materials, quality control procedures, and opinions regarding challenges existing within the community.

Results

A total of 125 laboratories participated in the questionnaire. A broad overview of results highlighted a wide methodological diversity within current lipidomic workflows. The impact of this diversity on lipid measurement and quantitation is currently unknown and needs to be explored further. While some laboratories do incorporate SOPs and quality control activities, these concepts have not been fully embraced by the community. The top five perceived challenges within the lipidomics community were a lack of standardization amongst methods/protocols, lack of lipid standards, software/data handling and quantification, and over-reporting/false positives.

Conclusion

The questionnaire provided an overview of current lipidomics methodologies and further promoted the need for community-accepted guidelines and protocols. The questionnaire also served as a platform to help determine and prioritize metrological issues to be investigated.

     

Direct quantitation of psychosine from alkaline-treated lipid extracts with a semi-synthetic internal standard

Psychosine is an important bioactive sphingolipid metabolite and plays an essential role in the pathogenesis of Krabbe's disease. Herein, we extended shotgun lipidomics for the characterization and quantitation of psychosine in alkaline-treated crude lipid extracts by using neutral loss scan of 180 amicro (i.e., galactose) in the positive-ion mode. Specifically, we semi-synthesized N,N-dimethyl psychosine and used it as an internal standard for quantitation of psychosine. After characterization of the fragmentation patterns of psychosine and the selected internal standard and optimization of the experimental conditions, we demonstrated that a broad linear dynamic range for the quantitation of psychosine and a limit of detection at a concentration of low fmol/microl were achieved using this approach. The developed method is generally simpler and more efficient than other previously reported methods. Multiple factors influencing quantitation of psychosine were extensively examined and/or discussed. The levels of psychosine in diabetic mouse nerve tissue samples were determined by the developed methodology. Collectively, the developed approach, as a new addition to the shotgun lipidomics technology, will be extremely useful for understanding the pathways/networks of sphingolipid metabolism and for exploring the important roles of psychosine in a variety of physiological and pathological conditions.     

Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach

Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO(3)](-)) in the negative-ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/mul concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies.     

Reporting of lipidomics data should be standardized

This article highlights, to our opinion, some of the most pertinent issues related to producing high quality lipidomics data. These issues include pitfalls related to sample collection and storage, lipid extraction, the use of shotgun and LC-MS-based lipidomics approaches, and the identification, annotation and quantification of lipid species. We hope that highlighting these issues will help stimulate efforts to implement reporting standards for dissemination of lipidomics data. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

Lipidomics: Prospects from a technological perspective

Over the last two decades, lipidomics has evolved into an ‘omics’ technology pari passu with benchmarking ‘omics’ technologies, such as genomics or proteomics. The driving force behind this development was a constant advance in mass spectrometry and related technologies. The aim of this opinion article is to give the interested reader a concise but still comprehensive overview about the technological state of the art in lipidomics, current challenges and perspectives for future development. As such, this article guides through the whole workflow of lipidomics, from sampling to data analysis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

Exploring the lipoprotein composition using Bayesian regression on serum lipidomic profiles

MOTIVATION: Serum lipids have been traditionally studied in the context of lipoprotein particles. Today's emerging lipidomics technologies afford sensitive detection of individual lipid molecular species, i.e. to a much greater detail than the scale of lipoproteins. However, such global serum lipidomic profiles do not inherently contain any information on where the detected lipid species are coming from. Since it is too laborious and time consuming to routinely perform serum fractionation and lipidomics analysis on each lipoprotein fraction separately, this presents a challenge for the interpretation of lipidomic profile data. An exciting and medically important new bioinformatics challenge today is therefore how to build on extensive knowledge of lipid metabolism at lipoprotein levels in order to develop better models and bioinformatics tools based on high-dimensional lipidomic data becoming available today. RESULTS: We developed a hierarchical Bayesian regression model to study lipidomic profiles in serum and in different lipoprotein classes. As a background data for the model building, we utilized lipidomic data for each of the lipoprotein fractions from 5 subjects with metabolic syndrome and 12 healthy controls. We clustered the lipid profiles and applied a regression model within each cluster separately. We found that the amount of a lipid in serum can be adequately described by the amounts of lipids in the lipoprotein classes. In addition to improved ability to interpret lipidomic data, we expect that our approach will also facilitate dynamic modelling of lipid metabolism at the individual molecular species level.     

Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization

This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions. Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes.     

脂质组学分析方法进展及其在癌症研究中的应用

脂质占人体内源性代谢物的一半以上,种类繁多,结构复杂,因而具有多种生物功能,与多种生命活动密切相关。脂质组学是代谢组学分支的新兴学科,它可以通过比较不同生理状态下脂质含量的变化,寻找代谢通路中关键的脂质生物标志物,最终揭示脂质在各种生命活动中的作用机制。随着质谱技术的进步,脂质组学在疾病脂类生物标志物的识别、疾病诊断、药物作用机制的研究等方面已展现出广泛的应用前景。本文主要就脂质组学近几年的分析方法进展及其在癌症中的最新应用进行了综述。     

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.     

A comprehensive method for lipid profiling by liquid chromatography-ion cyclotron resonance mass spectrometry

This work aims to combine chromatographic retention, high mass resolution and accuracy, MS/MS spectra, and a package for automated identification and quantitation of lipid species in one platform for lipidomic analysis. The instrumental setup elaborated comprises reversed-phase HPLC coupled to a Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT), and Lipid Data Analyzer (LDA) software. Data analysis for lipid species quantification in this platform is based on retention time, mass resolution of 200,000, and mass accuracy below 2 ppm. In addition, automatically generated MS/MS spectra provide structural information at molecular level. This LC/MS technology allows analyzing complex biological samples in a quantitative manner as shown here paradigmatically for murine lipid droplets having a huge surplus of triacylglycerol species. Chromatographic preseparation of the bulk lipid class alleviates the problem of ion suppression of lipid species from other classes. Extension of 1D to 2D chromatography is possible, yet time consuming. The platform affords unambiguous detection of lipid species as low as 0.1‰ within major lipid classes. Taken together, a novel lipidomic LC/MS platform based on chromatographic retention, high mass resolution and accuracy, MS/MS analysis, and quantitation software enables analysis of complex samples as demonstrated for lipid droplets.