Monitoring lipid alterations in Drosophila heads in an amyotrophic lateral sclerosis model with time-of-flight secondary ion mass spectrometry.

Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.

Elucidating the molecular landscape of the stratum corneum.

SignificanceSkin is recognized as an intricate assembly of molecular components, which facilitate cell signaling, metabolism, and protein synthesis mechanisms in order to offer protection, regulation, and sensation to the body. Our study takes significant steps to characterize in more detail the complex chemistry of the skin, in particular by generating a better understanding of the uppermost layer, the stratum corneum. Using a state-of-the-art 3D OrbiSIMS technique, we were able to observe the depth distribution, in situ, for a wide range of molecular species. This unprecedented molecular characterization of skin provides information that has the potential to benefit research into fundamental processes, such as those associated with skin aging and disease, and the development and delivery of effective topical formulations.

SIMS Analysis of Thin EUV Photoresist Films.

This study reports on the application of secondary ion mass spectrometry (SIMS) for examining thin (20-50 nm) chemically amplified resist films on silicon. SIMS depth profiling was carried out using a gas cluster ion beam to ensure minimal sputter-induced damage to the organic constituents of interest. Specific attention concerned the distribution of the photo acid generator (PAG) molecule within these films, along with the photo-induced fragmentation occurring on extreme ultra-violet photo exposure. Positive secondary ion spectra were collected using a traditional time of flight (ToF)-SIMS and the latest generation IONTOF Hybrid SIMS instrumentation equipped with an OrbitrapTM mass analyzer. Tandem mass spectrometry (MS/MS) capability within the OrbitrapTM secondary ion column was utilized to verify that the C19H17S+ secondary ion did indeed have the molecular structure consistent with the PAG structure. The superior mass resolving power of the OrbitrapTM mass analyzer (∼20× of the ToF mass analyzer) along with improved mass accuracy (a few ppm) proved pivotal in the mass spectral and depth profile analysis of these films. This was not the case for the ToF-SIMS experiments, as the mass spectra, as well as the associated depth profiles, exhibited severe molecular (isobaric) interferences.

Multimodal Imaging Mass Spectrometry to Identify Markers of Pulmonary Arterial Hypertension in Human Lung Tissue Using MALDI-ToF, ToF-SIMS, and Hybrid SIMS.

Pulmonary arterial hypertension (PAH) is a rare and deadly disease affecting roughly 15-60 people per million in Europe with a poorly understood pathology. There are currently no diagnostic tools for early detection nor does a curative treatment exist. The lipid composition of arteries in lung tissue samples from human PAH and control patients were investigated using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) combined with time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging. Using random forests as an IMS data analysis technique, it was possible to identify the ion at m/z 885.6 as a marker of PAH in human lung tissue. The m/z 885.6 ion intensity was shown to be significantly higher around diseased arteries and was confirmed to be a diacylglycerophosphoinositol PI(C18:0/C20:4) via MS/MS using a novel hybrid SIMS instrument. The discovery of a potential biomarker opens up new research avenues which may finally lead to a better understanding of the PAH pathology and highlights the vital role IMS can play in modern biomedical research.

New insights into ToF-SIMS imaging in osteoporotic bone research.

The present work focuses on the application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) in osteoporotic bone research. In order to demonstrate the benefit, the authors present concrete application examples of ToF-SIMS in three different areas of bone research. ToF-SIMS as a mass spectrometric imaging technique allows simultaneous visualization of mineralized and nonmineralized bone tissue as well as implanted biomaterials and bone implant interphases. In the first example, the authors show that it is possible to study the incorporation and distribution of different components released from bone filler materials into bone with a single mass spectrometric measurement. This not only enables imaging of nonstained bone cross sections but also provides further insights beyond histologically obtained information. Furthermore, they successfully identified several mass fragments as markers for newly formed cartilage tissue and growth joint in bone. Different modes of ToF-SIMS as well as different SIMS instruments (IONTOF's TOF.SIMS 5 and M6 Hybrid SIMS, Ionoptika's J105) were used to identify these mass signals and highlight the high versatility of this method. In the third part, bone structure of cortical rat bone was investigated from bone sections embedded in technovit (polymethyl methacrylate, PMMA) and compared to cryosections. In cortical bone, they were able to image different morphological features, e.g., concentric arrangement of collagen fibers in so-called osteons as well as Haversian canals and osteocytes. In summary, the study provides examples of application and shows the strength of ToF-SIMS as a promising analytical method in the field of osteoporotic bone research.

Imaging of Lipids in Native Human Bone Sections Using TOF-Secondary Ion Mass Spectrometry, Atmospheric Pressure Scanning Microprobe Matrix-Assisted Laser Desorption/Ionization Orbitrap Mass Spectrometry, and Orbitrap-Secondary Ion Mass Spectrometry.

A method is described for high-resolution label-free molecular imaging of human bone tissue. To preserve the lipid content and the heterogeneous structure of osseous tissue, 4 µm thick human bone sections were prepared via cryoembedding and tape-assisted cryosectioning, circumventing the application of organic solvents and a decalcification step. A protocol for comparative mass spectrometry imaging (MSI) on the same section was established for initial analysis with time-of-flight secondary ion mass spectrometry (TOF-SIMS) at a lateral resolution of 10 µm to <500 nm, followed by atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) Orbitrap MSI at a lateral resolution of 10 µm. This procedure ultimately enabled MSI of lipids, providing the lateral localization of major lipid classes such as glycero-, glycerophospho-, and sphingolipids. Additionally, the applicability of the recently emerged Orbitrap-TOF-SIMS hybrid system was exemplarily examined and compared to the before-mentioned MSI methods.

The 3D OrbiSIMS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power.

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 µm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

Analysis of Drosophila lipids by matrix-assisted laser desorption/ionization mass spectrometric imaging.

Drosophila melanogaster is a major model organism for numerous lipid-related diseases. While comprehensive lipidomic profiles have been generated for D. melanogaster, little information is available on the localization of individual lipid classes and species. Here, we show the use of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) to profile lipids in D. melanogaster tissue sections. The preparation of intact cryosections from whole insects presents a challenge due to the brittle hydrophobic cuticle surrounding the body and heterogeneous tissue types beneath the cuticle. However, the introduction of a novel sucrose infiltration step and gelatin as an embedding media greatly improved the quality of tissue sections. We generated MS image profiles of six major lipid classes: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and triacylglycerides. In addition, signals corresponding to two male-specific sex pheromones were detected in the ejaculatory bulb, a specialized site of pheromone production. MSI performed with 35 µm lateral resolution provided high sensitivity detection of at least 92 different lipid species, based on exact mass. In contrast, MSI with 10 µm lateral resolution enabled the detection of 36 lipid species but allowed lipid profiling of individual organs. The ability to localize lipid classes in intact sections from whole Drosophila provides a powerful tool for characterizing the effects of diet, age, stress, and environment on lipid production and distribution.

Analysis of free fatty acids by ultraviolet laser desorption ionization mass spectrometry using insect wings as hydrophobic sample substrates.

Physiologically relevant free fatty acids (FFAs) were analyzed by UV-laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (LDI-oTOF-MS). Dissected wings from Drosophila melanogaster fruit flies were used as the hydrophobic, laser energy strongly absorbing sample substrates. Using untreated substrates produces predominantly molecular [M + K](+) ions of the FFAs, whereas other alkali metal adducts can be generated by treating the wings with the corresponding alkali hydroxide before spotting of analyte. Limits of detection for the positive ion mode were determined for mixtures of isolated FFAs to values in the low 10 pmol range. Specific values depend on chain length and degree of unsaturation. R(2) coefficients for the analysis of saturated FFAs were found to be generally close to 0.98 over about 3 orders of magnitude if an internal standard (15:0 FFA) was added. Semiquantitative analyses of mixtures containing unsaturated FFAs are also possible but require more effort on the calibration strategy. Notably, both saturated and (poly-)unsaturated FFAs are detected sensitively in the presence of relatively high concentrations of other physiologically abundant lipids (phospholipids and triacyclglycerols). This simplifies screening of the FFA composition in crude tissue extracts. This feature is demonstrated by the analysis of a crude liver extract and that of fingermarks.

MALDI mass spectrometry imaging of bioactive lipids in mouse brain with a Synapt G2-S mass spectrometer operated at elevated pressure: improving the analytical sensitivity and the lateral resolution to ten micrometers.

Mass spectrometers from the Synapt-G1/G2 family (Waters) are widely employed for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). A lateral resolution of about 50 µm is typically achieved with these instruments, that is, however, below the often desired cellular resolution. Here, we show the first MALDI-MSI examples demonstrating a lateral resolution of about ten micrometers obtained with a Synapt G2-S HDMS mass spectrometer without oversampling. This improvement became possible by laser beam shaping using a 4:1 beam expander and a circular aperture for spatial mode filtering and by replacement of the default focusing lens. We used dithranol as an effective matrix for imaging of acidic lipids such as sulfatides, gangliosides, and phosphatidylinositols in the negative ion mode. At the same time, the matrix enables MS imaging of more basic lipids in the positive ion mode. Uniform matrix coatings with crystals having average dimensions between 0.5 and 3 µm were obtained upon spraying a chloroform/methanol matrix solution. Increasing the cooling gas pressure in the MALDI ion source after adding an additional gas line was furthermore found to increase the ion abundances of labile lipids such as gangliosides. The combined characteristics are demonstrated with the MALDI-MSI analysis of fine structures in coronal mouse brain slices.

Water ice is a soft matrix for the structural characterization of glycosaminoglycans by infrared matrix-assisted laser desorption/ionization.

Glycosaminoglycans (GAGs) are a class of heterogeneous, often highly sulfated glycans that form linear chains consisting of up to 100 monosaccharide building blocks and more. GAGs are ubiquitous constituents of connective tissue, cartilage, and the extracellular matrix, where they have key functions in many important biological processes. For their characterization by mass spectrometry (MS) and tandem MS, the high molecular weight polymers are usually enzymatically digested to oligomers with a low degree of polymerization (dp), typically disaccharides. However, owing to their lability elimination of sulfate groups upon desorption/ionization is often encountered leading to a loss of information on the analyte. Here, we demonstrate that, in particular, water ice constitutes an extremely mild matrix for the analysis of highly sulfated GAG disaccharides by infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Depending on the degree of sulfation, next to the singly charged ionic species doubly- and even triply charged ions are formed. An unambiguous assignment of the sulfation sites becomes possible by subjecting sodium adducts of the GAGs to low-energy collision-induced dissociation tandem MS. These ionic species exhibit a remarkable stability of the sulfate substituents, allowing the formation of fragment ions retaining their sulfation that arise from either cross-ring cleavages or rupture of the glycosidic bonds, thereby allowing an unambiguous assignment of the sulfation sites.

Progress in detection and structural characterization of glycosphingolipids in crude lipid extracts by enzymatic phospholipid disintegration combined with thin-layer chromatography immunodetection and IR-MALDI mass spectrometry.

In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS(1) and MS(2) analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics.

Infrared matrix-assisted laser desorption/ionization orthogonal-time-of-flight mass spectrometry employing a cooling stage and water ice as a matrix.

Although water ice has been utilized in the past as a matrix for infrared matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS), it has not found a wider use due to limitations in the analytical performance and technical demands on the employment of the necessary cooling stage. Here, we developed a temperature-controlled sample stage for use with an orthogonal time-of-flight mass spectrometer (MALDI-o-TOF-MS). The stage utilizes a combination of liquid nitrogen cooling and counterheating with a Peltier element. It allows adjustment of the sample temperature between ~-120 °C and room temperature. To identify optimal irradiation conditions for IR-MALDI with the water ice matrix, we first investigated the influence of excitation wavelength, varied between 2.7 and 3.1 µm, and laser fluence on the signal intensities of molecular substance P ions. These data suggest the involvement of transient melting of the ice during the laser pulse and primary energy deposition into liquid water. As a consequence, the best analytical performance is obtained at a wavelength corresponding to the absorption maximum of liquid water of about 2.94 µm. The current data significantly surpass the previously reported analytical features. The particular softness of the method is, for example, exemplified by the analysis of noncovalently bound holo-myoglobin and of ribonuclease B. This is also the first report demonstrating the analysis of an IgG monoclonal antibody (MW ~ 150 kDa) from a water ice matrix. Untypical for MALDI-MS, high charge states of multiply protonated species were moreover observed for some of the investigated peptides and even for lacto-N-fucopentaose II oligosaccharides. Using water ice as matrix is of particular interest for MALDI MS profiling and imaging applications since matrix-free spectra are produced. The MS and tandem MS analysis of metabolites directly from frozen food samples is demonstrated with the example of a strawberry fruit.

Direct laser desorption ionization of endogenous and exogenous compounds from insect cuticles: practical and methodologic aspects.

We recently demonstrated that ultraviolet laser desorption ionization orthogonal time-of-flight mass spectrometry (UV-LDI o-TOF MS) could be used for the matrix-free analysis of cuticular lipids (unsaturated aliphatic and oxygen-containing hydrocarbons and triacylglycerides) directly from individual Drosophila melanogaster fruit flies (Yew, J. Y.; Dreisewerd, K.; Luftmann, H.; Pohlentz, G.; Kravitz, E. A., Curr. Biol. 2009, 19, 1245-1254). In this report, we show that the cuticular hydrocarbon, fatty acid, and triglyceride profiles of other insects and spiders can also be directly analyzed from intact body parts. Mandibular pheromones from the jaw of a queen honey bee are provided as one example. In addition, we describe analytical features and examine mechanisms underlying the methodology. Molecular ions of lipids can be generated by direct UV-LDI when non-endogenous compounds are applied to insect wings or other body parts. Current sensitivity limits are in the 10 pmol range. We show also the dependence of ion signal intensity on collisional cooling gas pressure in the ion source, laser wavelength (varied between 280-380 nm and set to 2.94 µm for infrared LDI), and laser pulse energy.

Small colony variants of Staphylococcus aureus reveal distinct protein profiles.

Small-colony variants (SCVs) of Staphylococcus aureus represent a slow-growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild-type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin-induced SCV). Applying an ultra-high resolution chromatography and high mass accuracy MS(E) -based label-free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase-controlled manner covering early-exponential, late-exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down-regulation of the tricarboxylic acid (TCA) cycle-related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin-induced SCVs, the clinically derived SCVs showed no prominent up-regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up-regulation of TCA cycle-related parts, while oxidative stress-related proteins were down-regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain-interrupting mutants and their complemented counterparts.

Rapid metabolic profiling of Nicotiana tabacum defence responses against Phytophthora nicotianae using direct infrared laser desorption ionization mass spectrometry and principal component analysis.

BACKGROUND: Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection. RESULTS: Infrared laser desorption ionization orthogonal time-of-flight mass spectrometry allows rapid and simultaneous detection in both negative and positive ion mode of a wide range of naturally occurring primary and secondary metabolites. An unsupervised principal component analysis was employed to identify correlations between changes in metabolite expression (obtained at different times and sample treatment conditions) and the overall defence response.A one-dimensional projection of the principal components 1 and 2 obtained from positive ion mode spectra was used to generate a Biological Response Index (BRI). The BRI obtained for each sample treatment was compared with the number of dead cells found in the respective tissue. The high correlation between these two values suggested that the BRI provides a rapid assessment of the plant response against the pathogen infection. Evaluation of the loading plots of the principal components (1 and 2) reveals a correlation among three metabolic cascades and the defence response generated in infected leaves. Analysis of selected phytohormones by liquid chromatography electrospray ionization mass spectrometry verified our findings. CONCLUSION: The described methodology allows for rapid assessment of infection-specific changes in the plant metabolism, in particular of phenolics, alkaloids, oxylipins, and carbohydrates. Moreover, potential novel biomarkers can be detected and used to predict the quality of plant infections.

Field-based ion generation from microscale emitters on natural and artificial objects for atmospheric pressure mass spectrometry.

Field-based ion generation is described for ambient mass spectrometry. The technique allows the analysis of endogenously expressed chemicals and exogenously applied compounds directly from the cuticle of live insects in real time. Cuticular hairs serve as electric field-enhancing structures and play a key role in ion generation. Artificial emitters such as graphite whiskers or sharp metal tips replicate this effect.