Distribution of lipids in human brain

The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer’s disease.     

MALDI Mass Spectrometric Imaging of Lipids in Rat Brain Injury Models

Matrix-assisted laser desorption ionization/ionization imaging mass spectrometry (MALDI IMS) with a time-of-flight analyzer was used to characterize the distribution of lipid molecular species in the brain of rats in two injury models. Ischemia/reperfusion injury of the rat brain after bilateral occlusion of the carotid artery altered appearance of the phospholipids present in the hippocampal region, specifically the CA1 region. These brain regions also had a large increase in the ion abundance at m/z 548.5 and collisional activation supported identification of this ion as arising from ceramide (d18:1/18:0), a lipid known to be associated with cellular apoptosis. Traumatic brain injury model in the rat was examined by MALDI IMS and the area of damage also showed an increase in ceramide (d18:1/18:0) and a remarkable loss of signal for the potassium adduct of the most abundant phosphocholine molecular species 16:0/18:1 (PC) with a corresponding increase in the sodium adduct ion. This change in PC alkali attachment ion was suggested to be a result of edema and influx of extracellular fluid likely through a loss of Na/K-ATPase caused by the injury. These studies reveal the value of MALDI IMS to examine tissues for changes in lipid biochemistry and will provide data needed to eventually understand the biochemical mechanisms relevant to tissue injury.     

Selective imaging of positively charged polar and nonpolar lipids by optimizing matrix solution composition

Previous studies have shown that matrix‐assisted laser desorption/ionization–imaging mass spectrometry (MALDI‐IMS) is useful for studying the distribution of various small metabolites, particularly lipids. However, in this technique, selective ionization of the target molecules is imperative, particularly when analyzing small molecules. Since the sample clean‐up procedures available for the MALDI‐IMS of small metabolites are limited, the tissue sample will contain numerous molecular species other than the target molecules. These molecules will compete for ionization resulting in severe ion suppression. Hence, it is necessary to develop and optimize a sample preparation protocol for the target molecules. In this study, through model experiments using reference compounds, we optimized the composition of the matrix solution used for positively charged lipids in terms of the concentration of the organic solvent and presence/absence of alkali metal salts. We demonstrated that a high concentration of organic solvent in the matrix solution favors the preferential detection of lipids over peptides. The presence of alkali metal salts in the matrix solution was favorable for the detection of polar lipids, while a salt‐free matrix solution was suitable for the detection of nonpolar lipids. Furthermore, potassium salts added to the matrix solution caused merging of various lipid adducts (adducts with proton, sodium, and potassium) into one single potassiated species. Using the optimized protocols, we selectively analyzed phosphatidylcholine (PC) and triacylglycerol (TG) with different fatty acid compositions in a rat kidney section. Copyright © 2009 John Wiley & Sons, Ltd.     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

Molecular histology analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry using gold nanoparticles as matrix

Gold nanoparticles (AuNPs) were applied and optimized as matrix for matrix‐assisted laser desorption/ionization mass spectrometry analysis of animal tissues, and enabled histological analysis of animal tissues at molecular level by imaging mass spectrometry (IMS). AuNPs were coated on animal tissue in a solvent‐free manner via argon ion sputtering. Metabolites, including neurotransmitters, fatty acids and nucleobases, were directly detected from mouse brain tissue. Based on region‐specific chemical profiles, fine histological features of mouse brain tissue and heterogeneous regions of tumor tissue were both revealed. Copyright © 2011 John Wiley & Sons, Ltd.     

A new lipidomics approach by thin-layer chromatography-blot-matrix-assisted laser desorption/ionization imaging mass spectrometry for analyzing detailed patterns of phospholipid molecular species

Thin-layer chromatography (TLC) is a highly established convenient technique for lipid separation and partial characterization of neutral and acidic glycosphingolipids (GSLs) and phospholipids, in mixtures. Meanwhile, imaging mass spectrometry (IMS) is a promising tool for lipidomics. However, some lipid classes are detected more sensitively than others, which can lead to suppression effects when complex mixtures are analyzed. Therefore to analyze complex lipid mixtures, a precise separation into the individual lipid classes is necessary. Here we present our highly sensitive and convenient analytical technology that combines TLC and IMS, namely the TLC-Blot–MALDI-IMS method, to visualize whole lipids and individual molecular species with high sensitivity compared with common staining methods. This method allows for easy visualization of all lipids with a linear range of approximately one order of magnitude and precision <16% RSD, making it useful for differential display analysis of lipids.     

Visualization of acetylcholine distribution in central nervous system tissue sections by tandem imaging mass spectrometry

Metabolite distribution imaging via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundances of larger metabolite species, it is important to expand its application to smaller molecules, such as neurotransmitters. This study aimed to develop an IMS application to visualize neurotransmitter distribution in central nervous system tissue sections. Here, we raise two technical problems that must be resolved to achieve neurotransmitter imaging: (1) the lower concentrations of bioactive molecules, compared with those of membrane lipids, require higher sensitivity and/or signal-to-noise (S/N) ratios in signal detection, and (2) the molecular turnover of the neurotransmitters is rapid; thus, tissue preparation procedures should be performed carefully to minimize postmortem changes. We first evaluated intrinsic sensitivity and matrix interference using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS) to detect six neurotransmitters and chose acetylcholine (ACh) as a model for study. Next, we examined both single MS imaging and MS/MS imaging for ACh and found that via an ion transition from m/z 146 to m/z 87 in MS/MS imaging, ACh could be visualized with a high S/N ratio. Furthermore, we found that in situ freezing method of brain samples improved IMS data quality in terms of the number of effective pixels and the image contrast (i.e., the sensitivity and dynamic range). Therefore, by addressing the aforementioned problems, we demonstrated the tissue distribution of ACh, the most suitable molecular specimen for positive ion detection by IMS, to reveal its localization in central nervous system tissues.     

Direct analysis of lipids in mouse brain using electrospray droplet impact/SIMS

Electrospray droplet impact (EDI)/secondary ion mass spectrometry (SIMS) is a new desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from the atmospheric‐pressure electrospray are accelerated in vacuum by 10 kV and impact the sample deposited on the metal substrate. EDI/SIMS was shown to enhance intact molecular ion formation dramatically compared to conventional SIMS. EDI/SIMS has been successfully applied to the analysis of mouse brain without any sample preparation. Five types of lipids, i.e. phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), galactocerebroside (GC) and sulfatide (ST), were readily detected from mouse brain section. In addition, by EDI/SIMS, six different regions of the mouse brain (cerebral cortex, corpus callosum, striatum, medulla oblongata, cerebellar cortex and cerebellar medulla) were examined. While GCs and STs were found to be rich in white matter, PIs were rich in gray matter. Copyright © 2010 John Wiley & Sons, Ltd.     

Spatiotemporal alteration of phospholipids and prostaglandins in a rat model of spinal cord injury

We determined quantitative and qualitative alterations in lipids during the occurrence and progression of spinal cord injury (SCI) in rats to identify potential clinical indicators of SCI pathology. Imaging mass spectrometry (IMS) was used to visualize twelve molecular species of phosphatidylcholine (PC) on thin slices of spinal cord with SCI. In addition, twelve species of phospholipids and five species of prostaglandins (PGs) were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) of lipid extracts from control/injured spinal cords. Unique distribution patterns were observed for phospholipids with different fatty acid compositions, and distinct dynamic changes were seen in both their amounts and their distributions in tissue as tissue damage resulting from SCI progressed. In particular, PCs containing docosahexaenoic acid localized to the large nucleus in the anterior horn region at one day post-SCI and rapidly decreased thereafter. In contrast, PCs containing arachidonic acid (AA-PCs) were normally found in the posterior horn region and were intensely and temporarily elevated one week after SCI. Lysophosphatidylcholines (LPCs) also increased at the same SCI stage and in regions with elevated AA-PCs, indicating the release of AA and the production of PGs. Moreover, LC-ESI-MS/MS analysis of lipid extracts from the spinal cord tissue at the impact site demonstrated a peak in PGE2 that reflected the elevation/reduction pattern of AA-PCs and LPC. Although further investigation is required, we suggest that invasive immune cells that penetrated from the impaired blood-brain barrier at 1-2 weeks post-SCI may have produced LPCs, released AA from AA-PCs, and produced PGs in SCI tissue at sites enriched in AA-PCs/LPC.     

Imaging of Phospholipids in Formalin Fixed Rat Brain Sections by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a valuable tool for the analysis of molecules directly from tissue. Imaging of phospholipids is gaining widespread interest, particularly as these lipids have been implicated in a variety of pathologic processes. Formalin fixation (FF) is the standard protocol used in histology laboratories worldwide to preserve tissue for analysis, in order to aid in the diagnosis and prognosis of diseases. This study assesses MALDI imaging of phospholipids directly in formalin fixed tissue, with a view to future analysis of archival tissue. This investigation proves the viability of MALDI-MSI for studying the distribution of lipids directly in formalin fixed tissue, without any pretreatment protocols. High quality molecular images for several phosphatidylcholine (PC) and sphingomyelin (SM) species are presented. Images correspond well with previously published data for the analysis of lipids directly from freshly prepared tissue. Different ionization pathways are observed when analyzing fixed tissue compared with fresh, and this change was found to be associated with formalin buffers employed in fixation protocols. The ability to analyze lipids directly from formalin fixed tissue opens up new doors in the investigation of disease profiles. Pathologic specimens taken for histologic investigation can be analyzed by MALDI-MS to provide greater information on the involvement of lipids in diseased tissue.     

Direct tissue analysis of phospholipids in rat brain using MALDI-TOFMS and MALDI-ion mobility-TOFMS

After water, lipids are the most common biomolecules found in the brain (12%). A brief perusal of the physiology, anatomy, and pathophysiology of the brain illustrates the importance of lipids. Recent advances in mass spectrometry have allowed the direct probing of tissues. However, most studies have focused on proteins. In the present work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and MALDI-ion mobility (IM)-TOFMS were employed for direct analysis of phospholipids in rat brain tissue. Molecular ions (MH⁺) corresponding to phosphatidylcholines, phosphatidylethanolamines, and sphingomyelin, were recorded. When studying pharmacology, we learn that many therapeutic compounds are stored in the body’s adipose tissue. MALDI-TOFMS and MALDI- IM-TOFMS were thus used to analyze rat brain tissue with chlorisondamine added directly onto the tissue slice. With both techniques, noncovalent complexes between the tissue phospholipids and chlorisondamine were detected. In addition, MALDI-IM-TOFMS of noncovalent complexes between phospholipids and chlorisondamine displayed a mobility between that of an isobaric lipid and peptide.     

High-resolution MALDI imaging mass spectrometry allows localization of peptide distributions at cellular length scales in pituitary tissue sections

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been used to determine peptide distributions directly from rat, mouse and human pituitary tissue sections. Since these organs are small (10²–10³ μm) the spatial resolution of IMS is a key issue in molecular imaging of pituitary tissue sections. Here we show that high-resolution IMS allows localization of neuropeptide distributions within different cell clusters of a single organ of a pituitary tissue section. The sample preparation protocol does not result in analyte redistribution and is therefore applicable to IMS experiments at cellular length scales. The stigmatic imaging mass spectrometer used in this study produces selected-ion-count images with pixel sizes of 500 nm and a resolving power of 4 μm, yielding superior spatial detail compared to images obtained in microprobe imaging experiments. Furthermore, we show that with imaging mass spectrometry a distinction can be made between different mammalian tissue sections based on differences in the amino acid sequence of neuropeptides with the same function. This example demonstrates the power of IMS for label-free molecular imaging at relevant biological length scales.     

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry reveals a layered distribution of phospholipid molecular species in the mouse retina

We recently developed a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry (IMS) system. This system enables us to perform structural analyses using tandem mass spectrometry (MS/MS), as well as to visualize phospholipids and peptides in frozen sections. In the retina, phototransduction is regulated by the light-sensitive interaction between visual pigment-coupled receptor proteins, such as rhodopsin, and G proteins, such as transducin. There are some reports that the conformation of rhodopsin is influenced by the composition of phospholipids in the lipid bilayer membrane. However, these results were based on in vitro experiments and have not been analyzed in vivo. In this study, we visualized and identified phospholipids in mouse retinal sections with the MALDI-QIT-TOF-based IMS system. From a spectrum obtained by raster-scanned analysis of the sections, ions with high signal intensities were selected and analyzed by MS/MS. As a result, sixteen ions were identified as being from four diacyl-phosphatidylcholine (PC) species, i.e., PC (16:0/16:0), PC (16:0/18:1), PC (16:0/22:6), and PC (18:0/22:6), with different ion forms. The ion images revealed different distributions on the retinal sections: PC (16:0/18:1) was distributed in the inner nuclear layer and outer plexiform layer, PC (16:0/16:0) in the outer nuclear layer and inner segment, and both PC (16:0/22:6) and PC (18:0/22:6) in the outer segment and pigment epithelium. In conclusion, our in vivo IMS analyses demonstrated a three-zone distribution of PC species on the retinal sections. This approach may be useful for analyzing lipid changes and their contribution to phototransduction in the retina.     

Direct profiling of myelinated and demyelinated regions in mouse brain by imaging mass spectrometry

One of the newly developed imaging mass spectrometry (IMS) technologies utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to map proteins in thin tissue sections. In this study, we evaluated the power of MALDI IMS as we developed it in our (Bruker) MALDI TOF (Reflex IV) and TOF-TOF (Ultraflex II) systems to study myelin patterns in the mouse central nervous system under normal and pathological conditions. MALDI IMS was applied to assess myelin basic protein (MBP) isoform-specific profiles in different regions throughout the mouse brain. The distribution of ions of m/z 14,144 and 18,447 displayed a striking resemblance with white matter histology and were identified as MBP isoform 8 and 5, respectively. In addition, we demonstrated a significant reduction of the MBP-8 peak intensity upon MALDI IMS analysis of focal ethidium bromide-induced demyelinated brain areas. Our MS images were validated by immunohistochemistry using MBP antibodies. This study underscores the potential of MALDI IMS to study the contribution of MBP to demyelinating diseases.     

Separation and identification of neutral cereal lipids by normal phase high-performance liquid chromatography,using evaporative light-scattering and electrospray mass spectrometry for detection

A novel method was developed for the analysis of molecular species in neutral lipid classes, using separation by normal phase high-performance liquid chromatography, followed by detection by evaporative light-scattering and electrospray ionization tandem mass spectrometry. Monoacid standards, i.e. sterol esters, triacylglycerols, fatty acids, diacylglycerols, free sterols and monoacylglycerols, were separated to baseline on microbore 3 μm-silica gel columns. Complete or partial separation of molecular species in each lipid class permitted identification by automatic tandem mass spectrometry of ammonium adducts, produced via positive electrospray ionization. After optimization of the method, separation and identification of molecular species of various lipid classes was comprehensively tested by analysis of neutral lipids from the free lipid extract of maize flour.     

Monitoring time‐dependent degradation of phospholipids in sectioned tissues by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is useful for visualizing the localization of phospholipids on biological tissue surfaces creating great opportunities for IMS in lipidomic investigations. With advancements in IMS of lipids, there is a demand for large‐scale tissue studies necessitating stable, efficient and well‐defined sample handling procedures. Our work within this article shows the effects of different storage conditions on the phospholipid composition of sectioned tissues from mouse organs. We have taken serial sections from mouse brain, kidney and liver thaw mounted unto ITO‐coated glass slides and stored them under various conditions later analyzing them at fixed time points. A global decrease in phospholipid signal intensity is shown to occur and to be a function of time and temperature. Contrary to the global decrease, oxidized phospholipid and lysophospholipid species are found to increase within 2 h and 24 h, respectively, when mounted sections are kept at ambient room conditions. Imaging experiments reveal that degradation products increase globally across the tissue. Degradation is shown to be inhibited by cold temperatures, with sample integrity maintained up to a week after storage in ?80 °C freezer under N₂ atmosphere. Overall, the results demonstrate a timeline of the effects of lipid degradation specific to sectioned tissues and provide several lipid species which can serve as markers of degradation. Importantly, the timeline demonstrates oxidative sample degradation begins appearing within the normal timescale of IMS sample preparation of lipids (i.e. 1–2 h) and that long‐term degradation is global. Taken together, these results strengthen the notion that standardized procedures are required for phospholipid IMS of large sample sets, or in studies where many serial sections are prepared together but analyzed over time such as in 3‐D IMS reconstruction experiments. Copyright © 2014 John Wiley & Sons, Ltd.     

Lipid profiling using catalytic pyrolysis/metal oxide laser ionization-mass spectrometry

A procedure for lipid analysis using catalytic pyrolysis metal oxide laser ionization mass spectrometry (CP-MOLI MS) is described. When surface activated CaO is mixed with a lipid sample and analyzed using CP-MOLI MS, cleavage of lipids occurs by a reaction that resembles thermal hydrolysis methylation. CP-MOLI MS of monoacylglycerides (MAG), diacylglycerides (DAG), triacylglycerides (TAG), varied fatty acids on DAGs and TAGs, phospholipids, algae, and bacteria produced Ca adducts of fatty acid constituents from the respective molecular species without matrix background interference. CP-MOLI MS offers increased speed and a streamlined analysis in which intact lipids as well as their representative fatty acid constituents can be profiled on the same instrument.     

The detection of glycosphingolipids in brain tissue sections by imaging mass spectrometry using gold nanoparticles

Glycosphingolipids (GSLs) are amphiphilic molecules consisting of a hydrophilic carbohydrate chain and a hydrophobic ceramide moiety. They appear to be involved primarily in biological processes such as cell proliferation, differentiation, and signaling. To investigate the mechanism of brain function in more detail, a more highly sensitive method that would reveal the GSL distribution in the brain is required. In this report, we describe a simple and efficient method for mapping the distribution and localization of GSLs present in mouse brain sections using nanoparticle-assisted laser desorption/ionization imaging mass spectrometry (IMS). We have developed and tested gold nanoparticles (AuNPs) as a new matrix to maximize the detection of GSLs. A matrix of AuNPs modified with alkylamine was used to detect various GSLs, such as minor molecular species of sulfatides and gangliosides, in mouse brain sections; these GSLs were hardly detected using 2,5-dihydroxybenzoic acid (DHB), which is the conventional matrix for GSLs. We achieved approximately 20 times more sensitive detection of GSLs using AuNPs compared to a DHB matrix. We believe that our new approach using AuNPs in IMS could lead to a new strategy for analyzing basic biological mechanisms and several diseases through the distribution of minor GSLs.     

MALDI-ion mobility-TOFMS imaging of lipids in rat brain tissue

While maintaining anatomical integrity, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has allowed researchers to directly probe tissue, map the distribution of analytes and elucidate molecular structure with minimal preparation. MALDI-ion mobility (IM)-orthogonal time-of-flight mass spectrometry (oTOFMS) provides an advantage by initially separating different classes of biomolecules such as lipids, peptides, and nucleotides by their IM drift times prior to mass analysis. In the present work the distribution of phosphatidlycholine and cerebroside species was mapped from 16 microm thick coronal rat brain sections using MALDI-IM-oTOFMS. Furthermore, the use of gold nanoparticles as a matrix enables detection of cerebrosides, which although highly concentrated in brain tissue, are not easily observed as positive ions because of intense signals from lipids such as phosphatidlycholines and sphingomyelins.     

Three-dimensional visualization of protein expression in mouse brain structures using imaging mass spectrometry

We have developed a method to visualize matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) data aligned with optically determinable tissue structures in three dimensions. Details of the methodology are exemplified using the 3-D reconstruction of myelin basic protein (MBP) in the corpus callosum of a mouse brain. In this procedure, optical images obtained from serial coronal sections are first aligned to each other to reconstruct a surface of the corpus callosum from segmented contours of the aligned images. The MALDI IMS data are then coregistered to the optical images and superimposed into the surface to create the final 3-D visualization. Correlating proteomic data with anatomical structures provides a more comprehensive understanding of healthy and pathological brain functions, and holds promise to be utilized in more complex anatomical arrangements.