Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.

Biosynthesis and differential spatial distribution of the 3-deoxyanthocyanidins apigenidin and luteolinidin at the interface of a plant-cyanobacteria symbiosis exposed to cold.

Aquatic ferns of the genus Azolla (Azolla) form highly productive symbioses with filamentous cyanobacteria fixing N2 in their leaf cavities, Nostoc azollae. Stressed symbioses characteristically turn red due to 3-deoxyanthocyanidin (DA) accumulation, rare in angiosperms and of unknown function. To understand DA accumulation upon cold acclimation and recovery, we integrated laser-desorption-ionization mass-spectrometry-imaging (LDI-MSI), a new Azolla filiculoides genome-assembly and annotation, and dual RNA-sequencing into phenotypic analyses of the symbioses. Azolla sp. Anzali recovered even when cold-induced DA-accumulation was inhibited by abscisic acid. Cyanobacterial filaments generally disappeared upon cold acclimation and Nostoc azollae transcript profiles were unlike those of resting stages formed in cold-resistant sporocarps, yet filaments re-appeared in leaf cavities of newly formed green fronds upon cold-recovery. The high transcript accumulation upon cold acclimation of AfDFR1 encoding a flavanone 4-reductase active in vitro suggested that the enzyme of the first step in the DA-pathway may regulate accumulation of DAs in different tissues. However, LDI-MSI highlighted the necessity to describe metabolite accumulation beyond class assignments as individual DA and caffeoylquinic acid metabolites accumulated differentially. For example, luteolinidin accumulated in epithelial cells, including those lining the leaf cavity, supporting a role for the former in the symbiotic interaction during cold acclimation.

Deep Characterisation of the sn-Isomer Lipidome Using High-Throughput Data-Independent Acquisition and Ozone-Induced Dissociation.

In recent years there has been a significant interest in the development of innovative lipidomics techniques capable of resolving lipid isomers. To date, methods applied to resolving sn-isomers have resolved only a limited number of species. We report a workflow based on ozone-induced dissociation for untargeted characterisation of hundreds of sn-resolved glycerophospholipid isomers from biological extracts in under 20 min, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn-isomer pairs identified as compared to previous reports and reveals that sn-isomer populations are tightly regulated and significantly different between cell lines. The sensitivity of this method and potential for de novo molecular discovery is further demonstrated by the identification of unexpected lipids containing ultra-long monounsaturated acyl chains at the sn-1 position.

Time-Resolved Imaging of High Mass Proteins and Metastable Fragments Using Matrix-Assisted Laser Desorption/Ionization, Axial Time-of-Flight Mass Spectrometry, and TPX3CAM.

The Timepix (TPX) is a position- and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 106 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757-970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.

Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.

Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an ∼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Mass Spectrometry Imaging of Lipids Using MALDI Coupled with Plasma-Based Post-Ionization on a Trapped Ion Mobility Mass Spectrometer.

Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines an atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma postionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behavior of a variety of lipid classes in positive- and negative-ion modes, and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser postionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE), as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based postionization with ion mobility is presented, and we show the value of ion mobility for the resolution and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.

Mass spectrometry imaging of phosphatidylcholine metabolism in lungs administered with therapeutic surfactants and isotopic tracers.

Mass spectrometry imaging (MSI) visualizes molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labeling provided spatial analyses of phosphatidylcholine (PC) metabolism in mouse lungs. Dysregulated surfactant metabolism is central to many respiratory diseases. Metabolism and turnover of therapeutic pulmonary surfactants were imaged from distributions of intact and metabolic products of an added tracer, universally 13C-labeled dipalmitoyl PC (U13C-DPPC). The parenchymal distributions of newly synthesized PC species were also imaged from incorporations of methyl-D9-choline. This dual labeling strategy demonstrated both lack of inhibition of endogenous PC synthesis by exogenous surfactant and location of acyl chain remodeling processes acting on the U13C-DPPC-labeled surfactant, leading to formation of polyunsaturated PC lipids. This ability to visualize discrete metabolic events will greatly enhance our understanding of lipid metabolism in diverse tissues and has potential application to both clinical and experimental studies.

Mass Spectrometry Imaging of Lipids with Isomer Resolution Using High-Pressure Ozone-Induced Dissociation.

Mass spectrometry imaging (MSI) of lipids within tissues has significant potential for both biomolecular discovery and histopathological applications. Conventional MSI technologies are, however, challenged by the prevalence of phospholipid regioisomers that differ only in the location(s) of carbon-carbon double bonds and/or the relative position of fatty acyl attachment to the glycerol backbone (i.e., sn position). The inability to resolve isomeric lipids within imaging experiments masks underlying complexity, resulting in a critical loss of metabolic information. Herein, ozone-induced dissociation (OzID) is implemented on a mobility-enabled quadrupole time-of-flight (Q-TOF) mass spectrometer capable of matrix-assisted laser desorption/ionization (MALDI). Exploiting the ion mobility region in the Q-TOF, high number densities of ozone were accessed, leading to ∼1000-fold enhancement in the abundance of OzID product ions compared to earlier MALDI-OzID implementations. Translation of this uplift into imaging resulted in a 50-fold improvement in acquisition rate, facilitating large-area mapping with resolution of phospholipid isomers. Mapping isomer distributions across rat brain sections revealed distinct distributions of lipid isomer populations with region-specific associations of isomers differing in double bond and sn positions. Moreover, product ions arising from sequential ozone- and collision-induced dissociation enabled double bond assignments in unsaturated fatty acyl chains esterified at the noncanonical sn-1 position.

Automated, parallel mass spectrometry imaging and structural identification of lipids.

We report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.

Ultra-High Mass Resolving Power, Mass Accuracy, and Dynamic Range MALDI Mass Spectrometry Imaging by 21-T FT-ICR MS.

Detailed characterization of complex biological surfaces by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) requires instrumentation that is capable of high mass resolving power, mass accuracy, and dynamic range. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers the highest mass spectral performance for MALDI MSI experiments, and often reveals molecular features that are unresolved on lower performance instrumentation. Higher magnetic field strength improves all performance characteristics of FT-ICR; mass resolving power improves linearly, while mass accuracy and dynamic range improve quadratically with magnetic field strength. Here, MALDI MSI at 21T is demonstrated for the first time: mass resolving power in excess of 1 600 000 (at m/z 400), root-mean-square mass measurement accuracy below 100 ppb, and dynamic range per pixel over 500:1 were obtained from the direct analysis of biological tissue sections. Molecular features with m/z differences as small as 1.79 mDa were resolved and identified with high mass accuracy. These features allow for the separation and identification of lipids to the underlying structures of tissues. The unique molecular detail, accuracy, sensitivity, and dynamic range combined in a 21T MALDI FT-ICR MSI experiment enable researchers to visualize molecular structures in complex tissues that have remained hidden until now. The instrument described allows for future innovative, such as high-end studies to unravel the complexity of biological, geological, and engineered organic material surfaces with an unsurpassed detail.

Simultaneous Detection of Zinc and Its Pathway Metabolites Using MALDI MS Imaging of Prostate Tissue.

Levels of zinc, along with its mechanistically related metabolites citrate and aspartate, are widely reported as reduced in prostate cancer compared to healthy tissue and are therefore pointed out as potential cancer biomarkers. Previously, it has only been possible to analyze zinc and metabolites by separate detection methods. Through matrix-assisted laser desorption/ionization mass spectrometry imaging (MSI), we were for the first time able to demonstrate, in two different sample sets (n = 45 and n = 4), the simultaneous spatial detection of zinc, in the form of ZnCl3-, together with citrate, aspartate, and N-acetylaspartate on human prostate cancer tissues. The reliability of the ZnCl3- detection was validated by total zinc determination using laser ablation inductively coupled plasma MSI on adjacent serial tissue sections. Zinc, citrate, and aspartate were correlated with each other (range r = 0.46 to 0.74) and showed a significant reduction in cancer compared to non-cancer epithelium (p < 0.05, log2 fold change range: -0.423 to -0.987), while no significant difference between cancer and stroma tissue was found. Simultaneous spatial detection of zinc and its metabolites is not only a valuable tool for analyzing the role of zinc in prostate metabolism but might also provide a fast and simple method to detect zinc, citrate, and aspartate levels as a biomarker signature for prostate cancer diagnostics and prognostics.

Evaluation of lipid coverage and high spatial resolution MALDI-imaging capabilities of oversampling combined with laser post-ionisation.

Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualising the spatial locations of lipids in biological tissues. However, a major challenge in interpreting the biological significance of local lipid compositions and distributions detected using MALDI-MSI is the difficulty in associating spectra with cellular lipid metabolism within the tissue. By-and-large this is due to the typically limited spatial resolution of MALDI-MSI (30-100 µm) meaning individual spectra represent the average spectrum acquired from multiple adjacent cells, each potentially possessing a unique lipid composition and biological function. The use of oversampling is one promising approach to decrease the sampling area and improve the spatial resolution in MALDI-MSI, but it can suffer from a dramatically decreased sensitivity. In this work we overcome these challenges through the coupling of oversampling MALDI-MSI with laser post-ionisation (MALDI-2). We demonstrate the ability to acquire rich lipid spectra from pixels as small as 6 µm, equivalent to or smaller than the size of typical mammalian cells. Coupled with an approach for automated lipid identification, it is shown that MALDI-2 combined with oversampling at 6 µm pixel size can detect up to three times more lipids and many more lipid classes than even conventional MALDI at 20 µm resolution in the positive-ion mode. Applying this to mouse kidney and human brain tissue containing active multiple sclerosis lesions, where 74 and 147 unique lipids are identified, respectively, the localisation of lipid signals to individual tubuli within the kidney and lipid droplets with lesion-specific macrophages is demonstrated. Graphical abstract.

Host-based lipid inflammation drives pathogenesis in Francisella infection.

Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.

Enhanced Sensitivity Using MALDI Imaging Coupled with Laser Postionization (MALDI-2) for Pharmaceutical Research.

Visualizing the distributions of drugs and their metabolites is one of the key emerging application areas of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) within pharmaceutical research. The success of a given MALDI-MSI experiment is ultimately determined by the ionization efficiency of the compounds of interest, which in many cases are too low to enable detection at relevant concentrations. In this work we have taken steps to address this challenge via the first application of laser-postionisation coupled with MALDI (so-called MALDI-2) to the analysis and imaging of pharmaceutical compounds. We demonstrate that MALDI-2 increased the signal intensities for 7 out of the 10 drug compounds analyzed by up to 2 orders of magnitude compared to conventional MALDI analysis. This gain in sensitivity enabled the distributions of drug compounds in both human cartilage and dog liver tissue to be visualized using MALDI-2, whereas little-to-no signal from tissue was obtained using conventional MALDI. This work demonstrates the vast potential of MALDI-2-MSI in pharmaceutical research and drug development and provides a valuable tool to broaden the application areas of MSI. Finally, in an effort to understand the ionization mechanism, we provide the first evidence that the preferential formation of [M + H]+ ions with MALDI-2 has no obvious correlation with the gas-phase proton affinity values of the analyte molecules, suggesting, as with MALDI, the occurrence of complex and yet to be elucidated ionization phenomena.

Reshaping Lipid Biochemistry by Pushing Barriers in Structural Lipidomics.

Lipidomics is a rapidly growing field with numerous examples showing the importance of lipid molecules throughout biology. It has also shed light onto the vast and complex functions performed by many lipids that possess an immense diversity in molecular structures. Mass spectrometry (MS) is the tool of choice for analyzing lipids and has been the key catalyst driving the field forward. However, MS does not yet permit true molecular lipidomics wherein the identification and quantification of lipids having defined molecular structures can be routinely achieved. Here we describe recent advances in MS-based lipidomics that allow access to higher levels of molecular information in lipidomics experiments. These advances will form a key piece of the puzzle as the field moves towards systems characterization of lipids at the molecular level.

Simultaneous lipidomic and transcriptomic profiling in mouse brain punches of acute epileptic seizure model compared to controls.

In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospholipids, endocannabinoids, and arachidonic acid, at high spatial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dorsal hippocampus, ventral hippocampus, basolateral amygdala, and hypothalamus. This extraction method combined with LC/multiple reaction monitoring for lipid quantifi-cation and quantitative PCR for RNA investigation allows lipidomic and transcriptomic profiling from submilligram amounts of tissue, thus benefiting the time and animal costs for analysis and the data reliability due to prevention of biological variability between animal batches and/or tissue heterogeneity, as compared with profiling in distinct animal batches. Moreover, the method allows a higher extraction efficiency and integrity preservation for RNA, while allowing concurrently quantitative analysis of low and high abundant lipids. The method was applied for brain punches obtained 1 h after kainic acid-induced epileptic seizures in mice (n = 10) compared with controls (n = 10), and enabled the provision of valuable new insights into the subregional lipid and RNA changes with epilepsy, highlighting its potential as a new viable tool in quantitative neurobiology.

Digestion-Free Analysis of Peptides from 30-year-old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging.

Formalin-fixed neuroendocrine tissues from American cockroaches ( Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histological localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample preparation protocol rendering the endogenous peptides readily amenable to MSI analysis. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aqueous washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly soluble are therefore retained in-tissue with this protocol. The method is fully "top-down", that is, without laborious in situ enzymatic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepared P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally, in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 year old, we demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. Our findings put forward a useful method for digestion-free, high-throughput analysis of endogenous peptides from FFPE samples and offer the potential for reinvestigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.

Targeted Drug and Metabolite Imaging: Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

Mass spectrometry imaging (MSI) has proven to be a valuable tool for drug and metabolite imaging in pharmaceutical toxicology studies and can reveal, for example, accumulation of drug candidates in early drug development. However, the lack of sample cleanup and chromatographic separation can hamper the analysis due to isobaric interferences. Multiple reaction monitoring (MRM) uses unique precursor ion-product ion transitions to add specificity which leads to higher selectivity. Here, we present a targeted imaging platform where desorption electrospray ionization is combined with a triple quadrupole (QqQ) system to perform MRM imaging. The platform was applied to visualize (i) lipids in mouse brain tissue sections and (ii) a drug candidate and metabolite in canine liver tissue. All QqQ modes were investigated to show the increased detection time provided by MRM as well as the possibility to perform dual polarity imaging. This is very beneficial for lipid imaging because some phospholipid classes ionize in opposite polarity (e.g., phosphatidylcholine/sphingomyelin in positive ion mode and phosphatidylserine/phosphatidylethanolamine in negative ion mode). Drug and metabolite images were obtained to show its strength in drug distribution studies. Multiple MRM transitions were used to confirm the local presence and selective detection of pharmaceutical compounds.

Spatial Systems Lipidomics Reveals Nonalcoholic Fatty Liver Disease Heterogeneity.

Hepatocellular lipid accumulation characterizes nonalcoholic fatty liver disease (NAFLD). However, the types of lipids associated with disease progression are debated, as is the impact of their localization. Traditional lipidomics analysis using liver homogenates or plasma dilutes and averages lipid concentrations, and does not provide spatial information about lipid distribution. We aimed to characterize the distribution of specific lipid species related to NAFLD severity by performing label-free molecular analysis by mass spectrometry imaging (MSI). Fresh frozen liver biopsies from obese subjects undergoing bariatric surgery ( n = 23) with various degrees of NAFLD were cryosectioned and analyzed by matrix-assisted laser desorption/ionization (MALDI)-MSI. Molecular identification was verified by tandem MS. Tissue sections were histopathologically stained, annotated according to the Kleiner classification, and coregistered with the MSI data set. Lipid pathway analysis was performed and linked to local proteome networks. Spatially resolved lipid profiles showed pronounced differences between nonsteatotic and steatotic tissues. Lipid identification and network analyses revealed phosphatidylinositols and arachidonic acid metabolism in nonsteatotic regions, whereas low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) metabolism was associated with steatotic tissue. Supervised and unsupervised discriminant analysis using lipid based classifiers outperformed simulated analysis of liver tissue homogenates in predicting steatosis severity. We conclude that lipid composition of steatotic and nonsteatotic tissue is highly distinct, implying that spatial context is important for understanding the mechanisms of lipid accumulation in NAFLD. MSI combined with principal component-linear discriminant analysis linking lipid and protein pathways represents a novel tool enabling detailed, comprehensive studies of the heterogeneity of NAFLD.