Quantitative imaging mass spectrometry of renal sulfatides: validation by classical mass spectrometric methods

Owing to its capability of discriminating subtle mass-altering structural differences such as double bonds or elongated acyl chains, MALDI-based imaging MS (IMS) has emerged as a powerful technique for analysis of lipid distribution in tissue at moderate spatial resolution of about 50 μm. However, it is still unknown if MS¹-signals and ion intensity images correlate with the corresponding apparent lipid concentrations. Analyzing renal sulfated glycosphingolipids, sulfatides, we validate for the first time IMS-signal identities using corresponding sulfatide-deficient kidneys. To evaluate the extent of signal quenching effects interfering with lipid quantification, we surgically dissected the three major renal regions (papillae, medulla, and cortex) and systematically compared MALDI IMS of renal sulfatides with quantitative analyses of corresponding lipid extracts by on-target MALDI TOF-MS and by ultra-performance LC-ESI-(triple-quadrupole)tandem MS. Our results demonstrate a generally strong correlation (R² > 0.9) between the local relative sulfatide signal intensity in MALDI IMS and absolute sulfatide quantities determined by the other two methods. However, high concentrations of sulfatides in the papillae and medulla result in an up to 4-fold signal suppression. In conclusion, our study suggests that MALDI IMS is useful for semi-quantitative dissection of relative local changes of sulfatides and possibly other lipids in tissue.     

Characterisation of sphingolipids in the human lens by thin layer chromatography–desorption electrospray ionisation mass spectrometry

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation–mass spectrometry (DESI–MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.     

Metabolomic profiling of biomarkers of liver X receptor-induced toxicity in mouse liver tissue

Methods based on matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS), liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer (LC-MS) and gas chromatography-mass spectrometry (GC-MS) were used to investigate changes in the small molecule profiles of mouse liver in response to administration of an LXR agonist. Mice were treated with either 0.3 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg or 60 mg/kg of an LXR test compound or saline (control) once daily, over a 5 day period, to investigate the effects of the drug on metabolism in the liver. It was possible to detect triacylglycerol accumulation in the livers of animals treated with the drug, even at the lowest concentrations using, in the first instance, MALDI MS. There was also an increase in the relative degree of triacylglycerol saturation in the drug-treated samples. Changes in the profiles of phosphatidylcholine lipids were also observed. The changes in lipid profiles were also confirmed by LC-MS and GC-MS, the latter revealing a large increase in the level of the free fatty acid oleic acid (C18:1) in the treated samples. All of the changes were dose-related. Polar metabolites in the samples were analysed by hydrophilic interaction (HILIC) chromatography in combination with an LTQ-Orbitrap mass spectrometer. There were many changes in the metabolite profiles, some of which might simply be related to generalised toxicity. The clearest marker compounds, which showed very marked changes with dose, were methylglutaryl carnitine (MGC) and hydroxymethylglutaryl carnitine (HMGC). Another marker of some interest was uridine diphosphate N-acetylglucosamine (UNGA).     

Parameters affecting the accuracy of the MALDI-TOF MS determination of the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio as potential marker of spermatozoa quality

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is increasingly used to characterize (phospho)lipids. However, quantitative MALDI data are often questioned because ion suppression may occur if mixtures are analyzed. Therefore, relative (but no absolute) data are normally derived from the MALDI mass spectra of lipid mixtures. We are particularly interested in the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio because it seems to represent a suitable measure of the inflammatory activity. In this study, different parameters affecting the achievable accuracy of the MALDI-TOF MS determination of the PC/LPC ratio are compared. It will be shown that particularly the applied laser fluence as well as the used solvents influence the accuracies. Using artificial lipid mixtures it will be demonstrated that the PC/LPC ratio can be determined with an accuracy of about ±10% making the MALDI assay comparable to established methods. Finally, it will be shown that the optimized conditions are also useful to determine the PC/LPC ratios in human seminal plasma.     

GM1 locates to mature amyloid structures implicating a prominent role for glycolipid-protein interactions in Alzheimer pathology

While the molecular mechanisms underlying Alzheimer's disease (AD) remain largely unknown, abnormal accumulation and deposition of beta amyloid (Aβ) peptides into plaques has been proposed as a critical pathological process driving disease progression. Over the last years, neuronal lipid species have been implicated in biological mechanisms underlying amyloid plaque pathology. While these processes comprise genetic features along with lipid signaling as well as direct chemical interaction of lipid species with Aβ mono- and oligomers, more efforts are needed to spatially delineate the exact lipid-Aβ plaque interactions in the brain. Chemical imaging using mass spectrometry (MS) allows to probe the spatial distribution of lipids and peptides in complex biological tissues comprehensively and at high molecular specificity. As different imaging mass spectrometry (IMS) modalities provide comprehensive molecular and spatial information, we here describe a multimodal ToF-SIMS- and MALDI-based IMS strategy for probing lipid and Aβ peptide changes in a transgenic mouse model of AD (tgAPP_ArcSwe). Both techniques identified a general AD-associated depletion of cortical sulfatides, while multimodal MALDI IMS revealed plaque specific lipid as well as Aβ peptide isoforms. In addition, MALDI IMS analysis revealed chemical features associated with morphological heterogeneity of individual Aβ deposits. Here, an altered GM1 to GM2/GM3 ganglioside metabolism was observed in the diffuse periphery of plaques but not in the core region. This was accompanied by an enrichment of Aβ1–40arc peptide at the core of these deposits. Finally, a localization of arachidonic acid (AA) conjugated phosphatidylinositols (PI) and their corresponding degradation product, lyso-phosphatidylinositols (LPI) to the periphery of Aβ plaques was observed, indicating site specific macrophage activation and ganglioside processing.     

A rapid separation technique for overcoming suppression of triacylglycerols by phosphatidylcholine using MALDI-TOF MS

Phospholipids and triacylglycerols (TAGs) are important classes of lipids in biological systems. Rapid methods have been developed for their characterization in crude samples, including MALDI time-of-flight MS. For mixtures, MALDI often selectively shows only some components. For example, phosphatidylcholine (PC) suppresses detection of other lipids. Most rapid MS methods detect either TAGs or phospholipids but not both. Herein, we demonstrate a simple approach to rapidly screen mixtures containing multiple lipid classes. To validate this approach, reference lipids [PC, tripalmitin (PPP), and phosphatidyl-ethanolamine (PE)] and real samples (beef, egg yolk) were used. In a binary mixture with a strong suppressor (PC), PPP was greatly suppressed. After a simple separation, suppression was virtually eliminated. A mixture of nominally nonsuppressing lipids (PE and PPP) was not adversely affected by separation. Ground beef and egg yolk were used to demonstrate detection of known lipid compositions where other methods have missed one or more lipids or lipid classes. Separation was performed using solid phase extraction with a PrepSep florisil column. A 10 min separation allows rapid screening for lipids and changes in lipids. It is sufficient to clearly detect all lipids and overcome suppression effects in complex lipid mixtures.     

Spatial distribution of glycerophospholipids in the ocular lens

Knowledge of the spatial distribution of lipids in the intraocular lens is important for understanding the physiology and biochemistry of this unique tissue and for gaining a better insight into the mechanisms underlying diseases of the lens. Following our previous study showing the spatial distribution of sphingolipids in the porcine lens, the current study used ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) to provide the whole lipidome of porcine lens and these studies were supplemented by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) of the lens using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to determine the spatial distribution of glycerophospholipids. Altogether 172 lipid species were identified with high confidence and their concentration was determined. Sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines were the most abundant lipid classes. We then determined the spatial and concentration-dependent distributions of 20 phosphatidylcholines, 6 phosphatidylethanolamines, and 4 phosphatidic acids. Based on the planar molecular images of the lipids, we report the organization of fiber cell membranes within the ocular lens and suggest roles for these lipids in normal and diseased lenses.     

MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is a severe, monogenetically inherited kidney and liver disease. PCK rats carrying the orthologous mutant gene serve as a model of human disease, and alterations in lipid profiles in PCK rats suggest that defined subsets of lipids may be useful as molecular disease markers. Whereas MALDI protein imaging mass spectrometry (IMS) has become a promising tool for disease classification, widely applicable workflows that link MALDI lipid imaging and identification as well as structural characterization of candidate disease-classifying marker lipids are lacking. Here, we combine selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis (FDA) of imaging data sets for identification of candidate markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high-resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases, suggesting that they be molecular markers of the disease and that a combination of MALDI imaging with high-resolution MS methods and Fisher discriminant data analysis may be applicable for lipid marker discovery.     

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.     

Lipid Atlas of Keratinocytes and Betulin Effects on its Lipidome Profiled by Comprehensive UHPLC–MS/MS with Data Independent Acquisition Using Targeted Data Processing

Betulin is a pentacyclic triterpene with demonstrated healing properties in mid‐dermal wounds. A few earlier studies have provided insights into the wound healing effects on the molecular level. However, there are still questions left on the molecular targets of betulin. Therefore, a pharmacolipidomics analysis of betulin is undertaken in human immortalized keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. For this purpose, lipid extracts of keratinocytes treated with betulin and untreated controls are comprehensively analyzed by an untargeted UHPLC–ESI–QTOF‐MS/MS lipidomics profiling workflow using data‐independent acquisition. Targeted data processing allows the identification of 611 lipid species from 21 different lipid classes. Statistical analysis of the identified lipids shows significant changes in 440 lipid species that can be described as downregulation of cholesteryl esters and triacylglycerides and upregulation of glycerophospholipids, sphingolipids, and diacylglycerides. Additionally, some other signals corresponding to triterpenes are found in the betulin group and suggested that betulin is incorporated (in the membrane) and metabolized in keratinocytes.     

Metabolomic profile of hepatitis C virus-infected hepatocytes

Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 5'-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes.     

Levels of plasma sulfatides C18 : 0 and C24 : 1 correlate with disease status in relapsing–remitting multiple sclerosis

Multiple sclerosis (MS) is considered an autoimmune demyelinating disease of the CNS and myelin‐derived glycolipids are one of the targets of this autoimmune attack. In this study, we examined for the first time the plasma distribution of sulfatide isoforms. Sulfatides with long‐chain (C24 : 0 or C24 : 1) and short‐chain (C16 : 0 or C18 : 0) fatty acids were quantified in plasma of relapsing–remitting MS patients by ultra‐high‐performance liquid chromatography tandem mass spectrometry. We found that C18 : 0 and C24 : 1 sulfatide plasma levels positively correlated with the Expanded Disability Status Scale. C16/C18 : 0 and C16/C24 : 0 ratios also correlated with the age and the time since last relapse. Healthy women showed higher levels of C16 : 0 sulfatide than healthy men; however, this gender difference disappeared in MS patients. Our data underline the potential use of sulfatides as biomarkers in relapsing–remitting MS and points to a possible association with the higher susceptibility of women to develop MS.

     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Surface analysis of lipids by mass spectrometry: More than just imaging

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the “omics” age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.     

Oxidative changes of lipids monitored by MALDI MS

Oxidation processes of lipids are of paramount interest from many viewpoints. For instance, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by oxidation of lipids or free fatty acids. Additionally, many inflammatory diseases are accompanied by lipid oxidation and, therefore, oxidation products are also useful disease (bio)markers. Thus, there is also considerable interest in methods of (oxidized) lipid analysis.Nowadays, soft ionization mass spectrometric (MS) methods are regularly used to study oxidative lipid modifications due to their high sensitivities and the extreme mass resolution. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are increasing. This review aims to summarize the so far available data on MALDI analyses of oxidized lipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids under in vivo conditions, particularly the oxidation of (human) lipoproteins. It is not the aim of this review to praise MALDI as the “best” method but to provide a critical survey of the advantages and drawbacks of this method.     

Revealing serum lipidomic characteristics and potential lipid biomarkers in patients with POEMS syndrome

POEMS syndrome is a rare plasma cell dyscrasia with distinct lipid metabolism abnormalities at disease onset. However, the serum lipidomic characteristics in patients with POEMS syndrome were not investigated. The study performed an untargeted lipidome screening by liquid chromatography-tandem mass spectrometry (LS-MS/MS) in the pre- and post-treatment serum of 24 patients with POEMS syndrome, together with the serum of 24 paired healthy controls. Patients with POEMS syndrome had a distinct serum lipid composition compared with healthy controls, and a 3-lipid model had a predictive accuracy of 93.5% in distinguishing patients and healthy controls consisting of fatty acyl 17-oxo-20Z-hexacosenoic acid, phosphatidylcholine(16:0/18:1(9Z)) and sterol lipid 5b-pregnanediol. Four lipids including 17-oxo-20Z-hexacosenoic acid (r = 0.423, P = .040) were correlated with risk stratification, and 2 lipids including Cer(d18:0/13:0) were inversely related to serum vascular endothelial growth factor level (r=−0.465, P = .022). Eleven lipids were related to disease activity, including arachidonic acid which was inversely related and lysoPC(20:4) which was positively related. The study indicated a distinct lipid characteristic profile of patients with POEMS syndrome different from healthy controls and identified several lipids that may serve as potential diagnostic markers and monitors of therapeutic efficacy, as well as indicating potential metabolism pathways involved in the pathological process.     

Plasma lipid profiling in a large population-based cohort

We have performed plasma lipid profiling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 μl of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identified statistically significant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was significantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk.