Mass Spectrometry Imaging Of Lipids Research Articles

Method optimization for benchtop mass spectrometry imaging of lipids in Eisenia hortensis

Method optimization for benchtop mass spectrometry imaging of lipids in Eisenia hortensis

Expansion Strategy-Driven Micron-Level Resolution Mass Spectrometry Imaging of Lipids in Mouse Brain Tissue

Expansion Strategy-Driven Micron-Level Resolution Mass Spectrometry Imaging of Lipids in Mouse Brain Tissue

Spatial neurolipidomics-MALDI mass spectrometry imaging of lipids in brain pathologies.

Given the complexity of nervous tissues, understanding neurochemical pathophysiology puts high demands on bioanalytical techniques with respect to specificity and sensitivity. Mass spectrometry imaging (MSI) has evolved to become an important, biochemical imaging technology for spatial biology in biological and translational research. The technique facilitates comprehensive, sensitive elucidation of the spatial distribution patterns of drugs, lipids, peptides, and small proteins in situ. Matrix-assisted laser desorption ionization (MALDI)-based MSI is the dominating modality due to its broad applicability and fair compromise of selectivity, sensitivity price, throughput, and ease of use. This is particularly relevant for the analysis of spatial lipid patterns, where no other comparable spatial profiling tools are available. Understanding spatial lipid biology in nervous tissue is therefore a key and emerging application area of MSI research. The aim of this review is to give a concise guide through the MSI workflow for lipid imaging in central nervous system (CNS) tissues and essential parameters to consider while developing and optimizing MSI assays. Further, this review provides a broad overview of key developments and applications of MALDI MSI-based spatial neurolipidomics to map lipid dynamics in neuronal structures, ultimately contributing to a better understanding of neurodegenerative disease pathology.

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.

Low-melting point agarose as embedding medium for MALDI mass spectrometry imaging and laser-capture microdissection-based proteomics

The combination of MALDI mass spectrometry imaging, laser-capture microdissection, and quantitative proteomics allows the identification and characterization of molecularly distinct tissue compartments. Such workflows are typically performed using consecutive tissue sections, and so reliable sectioning and mounting of high-quality tissue sections is a prerequisite of such investigations. Embedding media facilitate the sectioning process but can introduce contaminants which may adversely affect either the mass spectrometry imaging or proteomics analyses. Seven low-temperature embedding media were tested in terms of embedding temperature and cutting performance. The two media that provided the best results (5% gelatin and 2% low-melting point agarose) were compared with non-embedded tissue by both MALDI mass spectrometry imaging of lipids and laser-capture microdissection followed by bottom-up proteomics. Two out of the seven tested media (5% gelatin and 2% low-melting point agarose) provided the best performances on terms of mechanical properties. These media allowed for low-temperature embedding and for the collection of high-quality consecutive sections. Comparisons with non-embedded tissues revealed that both embedding media had no discernable effect on proteomics analysis; 5% gelatin showed a light ion suppression effect in the MALDI mass spectrometry imaging experiments, 2% agarose performed similarly to the non-embedded tissue. 2% low-melting point agarose is proposed for tissue embedding in experiments involving MALDI mass spectrometry imaging of lipids and laser-capture microdissection, proteomics of consecutive tissue sections.

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 lipids in advanced human atherosclerotic plaque

Background and Aims : Lipids play a major role in atherosclerosis and are therefore potential biomarker targets. Investigating the spatial distribution of lipids in plaques can aid in understanding the role of lipids in local pathobiology, plaque progression and subsequent biomarker discovery. In contrast to histology, mass spectrometry imaging (MSI) can visualize the spatial distribution of a wide variety of lipid species in human carotid plaques.

MALDI Mass Spectrometry Imaging of Lipids on Free-Floating Brain Sections and Immunohistochemically Colocalized Markers of Neurodegeneration.

In mass spectrometry imaging (MSI), the essential steps in sample preparation include collection and storage. The most widely used preservation procedure for MSI consists in freezing samples and storing them at temperatures below -80°C. On the other hand, the most common method for preserving biological samples in clinical practice is their fixation in paraformaldehyde. The storage of free-floating sections is a particular type of the preservation of paraformaldehyde-fixed tissues that is used in immunohistochemistry. This chapter describes the approach of the multimodal imaging of free-floating brain sections using the MSI of lipids and the immunohistochemistry of neurodegeneration markers.

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.

Lipid mass spectrometry imaging and proteomic analysis of severe aortic stenosis.

Severe aortic stenosis (AS) is prevalent in adults ≥ 65 years, a significant cause of morbidity and mortality, with no medical therapy. Lipid and proteomic alterations of human AS tissue were determined using mass spectrometry imaging (MSI) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to understand histopathology, potential biomarkers of disease, and progression from non-calcified to calcified phenotype. A reproducible MSI method was developed using healthy murine aortic valves (n = 3) and subsequently applied to human AS (n = 2). Relative lipid levels were spatially mapped and associated with different microdomains. Proteomics for non-calcified and calcified microdomains were performed to ascertain differences in expression. Increased pro-osteogenic and inflammatory lysophosphatidylcholine (LPC) 16:0 and 18:0 were co-localized with calcified microdomains. Proteomics analysis identified differential patterns in calcified microdomains with high LPC and low cholesterol as compared to non-calcified microdomains with low LPC and high cholesterol. Calcified microdomains had higher levels of: apolipoproteins (Apo) B-100 (p < 0.001) and Apo A-IV (p < 0.001), complement C3 and C4-B (p < 0.001), C5 (p = 0.007), C8 beta chain (p = 0.013) and C9 (p = 0.010), antithrombotic proteins alpha-2-macroglobulin (p < 0.0001) and antithrombin III (p = 0.002), and higher anti-calcific fetuin-A (p = 0.02), while the osteoblast differentiating factor transgelin (p < 0.0001), extracellular matrix proteins versican, prolargin, and lumican ( p < 0.001) and regulator protein complement factor H (p < 0.001) were higher in non-calcified microdomains. A combined lipidomic and proteomic approach provided insight into factors potentially contributing to progression from non-calcified to calcific disease in severe AS. Additional studies of these candidates and protein networks could yield new targets for slowing progression of AS.

Imaging Isomers on a Biological Surface: A Review.

Mass spectrometry imaging is an imaging technology that allows the localization and identification of molecules on (biological) sample surfaces. Obtaining the localization of a compound in tissue is of great value in biological research. Yet, the identification of compounds remains a challenge. Mass spectrometry alone, even with high-mass resolution, cannot always distinguish between the subtle structural differences of isomeric compounds. This review discusses recent advances in mass spectrometry imaging of lipids, steroid hormones, amino acids and proteins that allow imaging with isomeric resolution. These improvements in detailed identification can give new insights into the local biological activity of isomers.

Mass Spectrometry Imaging of Lipids in Human Skin Disease Model Hidradenitis Suppurativa by Laser Desorption Ionization from Silicon Nanopost Arrays

Neutral lipids have been implicated in a host of potentially debilitating human diseases, such as heart disease, type-2 diabetes, and metabolic syndrome. Matrix-assisted laser desorption ionization (MALDI), the method-of-choice for mass spectrometry imaging (MSI), has led to remarkable success in imaging several lipid classes from biological tissue sections. However, due to ion suppression by phospholipids, MALDI has limited ability to efficiently ionize and image neutral lipids, such as triglycerides (TGs). To help overcome this obstacle, we have utilized silicon nanopost arrays (NAPA), a matrix-free laser desorption ionization (LDI) platform. Hidradenitis suppurativa (HS) is a chronic, recurrent inflammatory skin disease of the apocrine sweat glands. The ability of NAPA to efficiently ionize lipids is exploited in the analysis of human skin samples from sufferers of HS. Ionization by LDI from NAPA allows for the detection and imaging of a number of neutral lipid species, including TGs comprised of shorter, odd-chain fatty acids, which strongly suggests an increased bacterial load within the host tissue, as well as hexosylceramides (HexCers) and galabiosyl-/lactosylceramides that appear to be correlated with the presence of HS. Our results demonstrate that NAPA-LDI-MSI is capable of imaging and potentially differentiating healthy and diseased human skin tissues based on changes in detected neutral lipid composition.

Matrix-assisted ionisation in vacuum mass spectrometry and imaging on a modified quadrupole-quadrupole-time-of-flight mass spectrometer

Matrix-Assisted Ionisation in Vacuum (MAIV) is a new ionisation technique which ionises non-volatile compounds producing electrospray ionisation-like spectra. Its simple, matrix-assisted laser desorption/ionisation-like sample preparation allows for rapid analysis, with no requirement for external energy in the form of a laser or high voltage to produce ions. Ionisation occurs when the matrix (often 3-nitrobenzonitrile) is exposed to sub-ambient pressure. Here, the first use of this revolutionary new ionisation technique to image biological samples is reported. A commercial quadrupole-quadrupole-time-of-flight mass spectrometer was modified to incorporate control of the ion source pressure and a reduced sampling cone orifice diameter. In initial experiments, optimisation of source pressure and matrix composition was carried out to increase the longevity of ion formation. It was noted during these experiments that ion production was only observed when the sample was directly under the sampling cone. Optimisation of sample extraction into the MAIV matrix by the addition of 5% chloroform enabled MAIV mass spectrometry imaging of lipids in rat brain sections to be carried out in raster imaging mode. Modification of the size and position of the sampling cone improved the selectivity obtainable in these images. Although the quality of these initial images is relatively poor, work is underway to improve the spatial resolution by further modification of the ion source and progress is reported.

Mass spectrometry imaging of lipids: untargeted consensus spectra reveal spatial distributions in Niemann-Pick disease type C1

Mass spectrometry imaging (MSI) is a tool to rapidly map the spatial location of analytes without the need for tagging or a reporter system. Niemann-Pick disease type C1 (NPC1) is a neurodegenerative, lysosomal storage disorder characterized by accumulation of unesterified cholesterol and sphingolipids in the endo-lysosomal system. Here, we use MSI to visualize lipids including cholesterol in cerebellar brain tissue from the NPC1 symptomatic mouse model and unaffected controls. To complement the imaging studies, a data-processing pipeline was developed to generate consensus mass spectra, thereby using both technical and biological image replicates to assess differences. The consensus spectra are used to determine true differences in lipid relative abundance; lipid distributions can be determined in an unbiased fashion without prior knowledge of location. We show the cerebellar distribution of gangliosides GM1, GM2, and GM3, including variants of lipid chain length. We also performed MALDI-MSI of cholesterol. Further analysis of lobules IV/V and X of the cerebellum gangliosides indicates regional differences. The specificity achieved highlights the power of MSI, and this new workflow demonstrates a universal approach for addressing reproducibility in imaging experiments applied to NPC1.

Faster raster matrix-assisted laser desorption/ionization mass spectrometry imaging of lipids at high lateral resolution

The clinical use of mass spectrometry imaging (MSI) is rapidly growing, and applications are expanding. Crucially, the analysis of clinical cohorts requires a combination of higher analytical throughput and higher lateral resolution. Here, we demonstrate the benefits of an improved design of a prototype MALDI source (referred as “uMALDI”) mounted on a SYNAPT HDMS G2-Si mass spectrometer – widely used by the MSI community. The uMALDI source allows for imaging of lipids on histologically well documented rat brain sections at a lateral resolution of 15 μm without oversampling. We also investigate the fast rastering capabilities of the platform enabled by a new web-based MS control program (i.e. “WREnS” Waters Research Enabled Software). The combination of the uMALDI and WREnS allows high spatial resolution images at acquisition rates up to 20 pixels per second. Additionally, we demonstrate the capability of this platform to characterize the lipid distribution of osteoarthritic (OA) cartilage at the cellular level, which highlights potential benefit for broader application in clinical context. Finally, we prove that ion mobility separation of isobaric lipid species is maintained and images can be acquired at a rate of 10 scans per seconds. This renders a unique ion mobility mass spectrometric imaging platform with high acquisition rates and high spatial resolution.

Assessment of pathological response to therapy using lipid mass spectrometry imaging.

In many cancers, the establishment of a patient’s future treatment regime often relies on histopathological assessment of tumor tissue specimens in order to determine the extent of the ‘pathological response’ to a given therapy. However, histopathological assessment of pathological response remains subjective. Here we use MALDI mass spectrometry imaging to generate lipid signatures from colorectal cancer liver metastasis specimens resected from patients preoperatively treated with chemotherapy. Using these signatures we obtained a unique pathological response score that correlates with prognosis. In addition, we identify single lipid moieties that are overexpressed in different histopathological features of the tumor, which have potential as new biomarkers for assessing response to therapy. These data show that computational methods, focusing on the lipidome, can be used to determine prognostic markers for response to chemotherapy and may potentially improve risk assessment and patient care.

Mass spectrometry imaging of lipids and metabolites: from fruit fly brains to single nanometer vesicles

Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article “Identification of proteins that are differentially expressed in brains with Alzheimer’s disease using iTRAQ labeling and tandem mass spectrometry” (Minjarez et al., 2016) [1].

MALDI Mass Spectrometry Imaging of Lipids and Gene Expression Reveals Differences in Fatty Acid Metabolism between Follicular Compartments in Porcine Ovaries.

In mammals, oocytes develop inside the ovarian follicles; this process is strongly supported by the surrounding follicular environment consisting of cumulus, granulosa and theca cells, and follicular fluid. In the antral follicle, the final stages of oogenesis require large amounts of energy that is produced by follicular cells from substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring oocyte developmental competence, the aim of this study was to investigate site-specificity of lipid metabolism in ovaries by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. Using MALDI Mass Spectrometry Imaging, images of porcine ovary sections were reconstructed from lipid ion signals for the first time. Cluster analysis of ion spectra revealed differences in spatial distribution of lipid species among ovarian compartments, notably between the follicles and interstitial tissue. Inside the follicles analysis differentiated follicular fluid, granulosa, theca and the oocyte-cumulus complex. Moreover, by transcript quantification using real time PCR, we showed that expression of five key genes in FA metabolism significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte. In conclusion, lipid metabolism differs between ovarian and follicular compartments.

MALDI mass spectrometry imaging of lipids and primary metabolites on rat brain sections.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) enables the localization and the structural identification of a large set of molecules on a surface with 10-20 μm resolution. MALDI is often coupled to time-of-flight (TOF) tandem analyzers which remain a versatile instrument for the detection and the structural analysis of molecular ions. This technique can be used to locate either large biomolecules, such as peptides/proteins, or small endogenous or exogenous ones. Among them, lipids and primary metabolites are of high interest because they can reflect the cell state. This chapter is thus dedicated to the analysis, on rat brain tissue sections, of lipids in positive and negative ion modes, and primary metabolites in the negative ion mode. A particular attention is paid to the structural characterization of lipids using lithium cationization in the positive ion mode.

Lipidomics

Lipidomics characterizes the composition of intact lipid molecular species in biological systems and the field has been driven by some spectacular advances in mass spectrometry instrumentation and applications. This review will highlight these advances and outline their recent application to address clinical issues. This review first identifies recent advances in lipid detection and analysis by a variety of mass spectrometry techniques, then reviews specific application including stable isotope labelling of lipids, lipid mass spectrometry imaging, data analysis and bioinformatics, and finally presents examples of the application of lipidomics to selected disease states. Lipidomics so far has been principally concerned with identifying novel methodologies, but recent advances demonstrating applications in diabetes, neurodegenerative diseases, cystic fibrosis and other respiratory diseases clearly indicate the potential usefulness of lipidomics both to generate biomarkers of disease and to probe signalling and metabolic processes.