MALDI-MSI Analysis Research Articles

Single Cell MALDI-MSI Analysis of Lipids and Proteins within a Replicative Senescence Fibroblast Model.

In this study, we evaluate lipids and select proteins in human lung fibroblasts (hLFs) to interrogate changes occurring due to aging and senescence. To study single cell populations, a comparison of cells adhered onto slides using poly-d-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-d-lysine approach was then utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-immunohistochemistry (IHC) platform for single-cell-level analyses. Two protein markers of senescence, vimentin and p21, were both observed within the fibroblast populations and quantified. Lipidomic analysis of the fibroblasts found 12 lipids significantly altered because of replicative senescence, including fatty acids, such as stearic acid, and ceramide phosphoethanolamine species (CerPE). Similar to previous reports, alterations were detected in putative fatty acid building blocks, ceramides, among other lipid species. Altogether, our results reveal the ability to detect lipids implicated in senescence and show alterations to protein expression between normal and senescent fibroblast populations, including differences between young and aged cells. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level to analyze both protein expression and lipid profiles in cultured cells, with a particular focus on changes associated with aging and senescence.

Single Cell MALDI-MSI Analysis of Lipids and Proteins within a Replicative Senescence Fibroblast Model.

In this study, we evaluate lipids and select proteins in human lung fibroblasts (hLFs) to interrogate changes occurring due to aging and senescence. To study single cell populations, a comparison of cells adhered onto slides using poly-D-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-D-lysine approach was than utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-immunohistochemistry (IHC) platform for single-cell level analyses. Two protein markers of senescence, vimentin and p21, were both observed within the fibroblast populations and quantified. Lipidomic analysis of the fibroblasts found twelve lipids significantly altered because of replicative senescence, including fatty acids, such as stearic acid, and ceramide phosphoethanolamine species (CerPE). Similar to previous reports, alterations were detected in putative fatty acid building blocks, ceramides, among other lipid species. Altogether, our results reveal the ability to detect lipids implicated in senescence and show alterations to protein expression between normal and senescent fibroblast populations, including differences between young and aged cells. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level to analyze both protein expression and lipid profiles in cultured cells, with a particular focus on changes associated with aging and senescence.

Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing.

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids' quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6-8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6-8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond.

Rhodiola crenulata alleviates hypobaric hypoxia-induced brain injury by maintaining BBB integrity and balancing energy metabolism dysfunction

Background/Purpose: Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba (R. crenulate), a famous and characteristic Tibetan medicine, has been demonstrated to exert an outstanding brain protection role in the treatment of high-altitude hypoxia disease. However, the metabolic effects of R. crenulate on high-altitude hypoxic brain injury (HHBI) are still incompletely understood. Herein, the anti-hypoxic effect and associated mechanisms of R. crenulate were explored through both in vivo and in vitro experiments. Study design/MethodsThe mice model of HHBI was established using an animal hypobaric and hypoxic chamber. R. crenulate extract (RCE, 0.5, 1.0 and 2.0 g/kg) and salidroside (Sal, 25, 50 and 100 mg/kg) was given by gavage for 7 days. Pathological changes and neuronal apoptosis of mice hippocampus and cortex were evaluated using H&E and TUNEL staining, respectively. The effects of RCE and Sal on the permeability of blood brain barrier (BBB) were detected by Evans blue staining and NIR-II fluorescence imaging. Meanwhile, the ultrastructural BBB and cerebrovascular damages were observed using a transmission electron microscope (TEM). The levels of tight junction proteins Claudin-1, ZO-1 and occludin were detected by immunofluorescence. Additionally, the metabolites in mice serum and brain were determined using UHPLC-MS and MALDI-MSI analysis. The cell viability of Sal on hypoxic HT22 cells induced by CoCl2 was investigated by cell counting kit-8. The contents of LDH, MDA, SOD, GSH-PX and SDH were detected by using commercial biochemical kits. Meanwhile, intracellular ROS, Ca2+ and mitochondrial membrane potential were determined by corresponding specific labeled probes. The intracellular metabolites of HT22 cells were performed by the targeted metabolomics analysis of the Q300 kit. The cell apoptosis and necrosis were examined by YO-PRO-1/PI, Annexin V/PI and TUNEL staining. In addition, mitochondrial morphology was tested by Mito-tracker red with confocal microscopy and TEM. Real-time ATP production, oxygen consumption rate, and proton efflux rate were measured using a Seahorse analyzer. Subsequently, MCU, OPA1, p-Drp1ser616, p-AMPKα, p-AMPKβ and Sirt1 were determined by immunofluorescent and western blot analyses. ResultsThe results demonstrated that R. crenulate and Sal exert anti-hypoxic brain protection from inhibiting neuronal apoptosis, maintaining BBB integrity, increasing tight junction protein Claudin-1, ZO-1 and occludin and improving mitochondrial morphology and function. Mechanistically, R. crenulate and Sal alleviated HHBI by enhancing the tricarboxylic acid cycle to meet the demand of energy of brain. Additionally, experiments in vitro confirmed that Sal could ameliorate the apoptosis of HT22 cells, improve mitochondrial morphology and energy metabolism by enhancing mitochondrial respiration and glycolysis. Meanwhile, Sal-mediated MCU inhibited the activation of Drp1 and enhanced the expression of OPA1 to maintain mitochondrial homeostasis, as well as activation of AMPK and Sirt1 to enhance ATP production. ConclusionCollectively, the findings suggested that RCE and Sal may afford a protective intervention in HHBI through maintaining BBB integrity and improving energy metabolism via balancing MCU-mediated mitochondrial homeostasis by activating the AMPK/Sirt1 signaling pathway.

MALDI MS Imaging of Cucumbers.

There are many different methodologies in the literature for the preparation of plant material for subsequent MALDI MSI analysis. This chapter overviews preparation of cucumbers (Cucumis sativus L.), with emphasis on sample freezing, cryosectioning, and matrix deposition. This should act as a representative example of sample preparation for plant tissue, and due to wide sample variation (e.g., leaves, seeds, and fruit) and analytes of interest, method optimization will be required for different samples.

Quantitative MALDI mass spectrometry imaging for exploring cutaneous drug delivery of tofacitinib in human skin

In skin penetration studies, HPLC-MS/MS analysis on extracts of heat-separated epidermis and dermis provides an estimate of the amount of drug penetrated. In this study, MALDI-MSI enabled qualitative skin distribution analysis of endogenous molecules and the drug molecule, tofacitinib and quantitative analysis of the amount of tofacitinib in the epidermis. The delivery of tofacitinib to the skin was investigated in a Franz diffusion cell using three different formulations (two oil-in-water creams, C1 and C2 and an aqueous gel). Further, in vitro release testing (IVRT) was performed and resulted in the fastest release of tofacitinib from the aqueous gel and the lowest from C2. In the ex vivo skin penetration and permeation study, C1 showed the largest skin retention of tofacitinib, whereas, lower retention and higher permeation were observed for the gel and C2. The quantitative MALDI-MSI analysis showed that the content of tofacitinib in the epidermis for the C1 treated samples was comparable to HPLC-MS/MS analysis, whereas, the samples treated with C2 and the aqueous gel were below LOQ. The study demonstrates that MALDI-MSI can be used for the quantitative determination of drug penetration in epidermis, as well as, to provide valuable information on qualitative skin distribution of tofacitinib.

P0354MATRIX ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY IN PROTEIN ALTERATIONS ASSOCIATED WITH THE PROGRESSION OF IGA NEPHROPATHY DISCOVERY

Abstract Background and Aims IgA nephropathy (IgAN) is one of the most diffuse glomerulonephrites worldwide, but there are still many issues regarding its prognosis and pathogenesis understanding. Although the diagnosis is established by renal biopsy examination, there are still remaining pitfalls in primary origin discrimination, and therefore for prognosis and outcome for the patient. Method In this pilot study we performed matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) with a high spatial resolution (raster sampling of 20 μm) on formalin fixed paraffin embedded renal biopsies obtained from patients with IgAN (n=11) and other mesangioproliferative glomerulonephrites (MesPGN, n=6) attempting to enlighten proteomic alterations that may be associated with the progression of IgAN. The patients in both disease groups were separated according to their CKD stage (CKDI, CKDII, CKDIII and more). After MALDI-MSI analysis, all biopsies were stained with hematoxylin and eosin, scanned and overlaid with molecular image in order to verify the signals’ spatial distribution. Results Using MALDI-MSI we detected clear differences in the proteomic profiles of IgAN and other MesPGN tissues. Fourteen signals (AUC ≥ 0.8) were observed to have an altered intensity among the different CKD stages within the IgAN group. In particular, large increases in the intensity of these signals could be observed at CKD stages II and above. Putatively identified, these signals primarily corresponded to proteins involved in inflammatory and healing pathways and their increased intensity was localised within regions of tissue with large amounts of inflammatory cells or sclerosis (Figure 1), verified by immunohistochemical staining (Figure 2). Conclusion The capability of MALDI-MSI to provide highly spatially resolved proteomics analysis of complex renal tissue demonstrates it a useful additional diagnostic and prognostic tool and a promising approach in the search for prognostic or predictive markers in glomerular diseases.

Novel Blotting Method for Mass Spectrometry Imaging of Metabolites in Strawberry Fruit by Desorption/Ionization Using Through Hole Alumina Membrane.

Mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization (MALDI) is a powerful technique for visualizing metabolites in the strawberry fruit. During sample preparation for MALDI-MSI, sectioning of the samples is usually required. In general, MALDI-MSI analysis of strawberry fruits that are larger than a single glass slide is difficult because thin sections cannot be prepared. In this study, we attempted to visualize metabolites in large strawberry fruits by MSI, employing a blotting method that uses desorption ionization using a through-hole alumina membrane (DIUTHAME) chip. Large strawberry fruits were cut and a DIUTHAME chip was set on the cross-section to blot the metabolites. After drying the DIUTHAME chip, the metabolites were measured in positive and negative ion modes using a commercial MALDI-type mass spectrometer. Several peaks were detected in both the ion modes. Various metabolites related to food quality, such as sugars, organic acids, and anthocyanins, were detected and successfully visualized by blotting on a DIUTHAME chip in MSI. These results suggest that blotting using a DIUTHAME chip in MSI is useful for visualizing the metabolites present in the strawberry fruit.

Unique Distribution of Diacyl-, Alkylacyl-, and Alkenylacyl-Phosphatidylcholine Species Visualized in Pork Chop Tissues by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Phosphatidylcholine (PC) is the major phospholipid in meat and influences meat qualities, such as healthiness. PC is classified into three groups based on the bond at the sn-1 position: Diacyl, alkylacyl, and alkenylacyl. To investigate their composition and distribution in pork tissues, including longissimus thoracis et lumborum (loin) spinalis muscles, intermuscular fat, and transparent tissues, we performed matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI). Eleven diacyl-, seven alkylacyl-, and six alkenylacyl-PCs were identified using liquid chromatography (LC)-tandem MS (MS/MS) analysis. Despite many alkylacyl- and alkenylacyl-PC species sharing identical m/z values, we were able to visualize these PC species using MALDI–MSI. Diacyl- and alkylacyl- and/or alkenylacyl-PC species showed unique distribution patterns in the tissues, suggesting that their distribution patterns were dependent on their fatty acid compositions. PCs are a major dietary source of choline in meat, and the amount was significantly higher in the muscle tissues. Consumption of choline mitigates age-related memory decline and neurodegenerative diseases; therefore, the consumption of pork muscle tissues could help to mitigate these diseases. These results support the use of MALDI–MSI analysis for assessing the association between PC species and the quality parameters of meat.

Latent Fingerprint Enhancement Techniques: A Review

Fingerprint (FP) is a global mark used for personal identification. This study reviews recent latent fingerprint (LFP) enhancement techniques including metal oxides, multi-metal deposition (MMD-I/II/SMD/Au-ASP), optical, chemical, physical, and physicochemical. Furthermore, analytical techniques involved in identification, evaluation, and determination of pieces of evidence, and perpetrators including the SEM, TEM, UV-Vis, IR/NIR, SERS, SKP, DLS, MALDI-MSI, and TD analysis were also discussed. Among numerous LFP enhancement techniques, the application of chemical methods in combination with optical techniques has a greater place to recover FPs with sufficient quality. However, instead of using such a complex and costly enhancing agent, nowadays nanotechnology is using specific techniques used for visualizing, inspecting, gathering, and analyzing trace evidence at the scene of a crime. To indicate using simple metal oxides such as ZnO that have superior fluorescent properties and also that consider both the surface and cost of the materials, enhancement of the LFP is functioning.

Mass spectrometry imaging guided molecular networking to expedite discovery and structural analysis of agarwood natural products

Structural analysis of biomolecules is essential to natural product discovery, especially for precious biomaterials such as agarwood. However, one of the greatest challenges to the characterization of natural products is the profound cost in time and manpower to the structural elucidation of these highly diverse compounds. Here, we demonstrate a multi-modal mass spectrometric strategy, integrating matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) and mass spectral molecular networking, to uncover agarwood natural products of Aquilaria sinensis trees. A simple workflow for preparing wood sections for MALDI-MSI analysis was demonstrated. Notably, tens of natural products in the agarwood region in wood stem section of A. sinensis were spatially revealed by MALDI-MSI. For the first time, such a great number of plant specialized metabolites is obtained by a single wood section MSI. Guided by the spatially resolved features, mass spectral molecular networking was subsequently applied for structural analysis of the agarwood natural products, in which three major classes of 2-(2-phenylethyl)chromones and their analogues were putatively characterized. These results suggest an efficient strategy to the dereplication of plant natural products.

The management of haemoglobin interference for the MALDI-MSI proteomics analysis of thyroid fine needle aspiration biopsies.

MALDI-MSI represents an ideal tool to explore the spatial distribution of proteins directly in situ, integrating molecular and cytomorphological information, enabling the discovery of potential diagnostic markers in thyroid cytopathology. However, red cells present in the fine needle aspiration biopsy (FNAB) specimens caused ion suppression of other proteins during the MALDI-MSI analysis due to large amount of haemoglobin. Aim of this study was to set up a sample preparation workflow able to manage this haemoglobin interference. Three protocols were compared using ex vivo cytological samples collected from fresh thyroid nodules of 9 patients who underwent thyroidectomy: (A) conventional air-dried smears, (B) cytological smears immediately fixed in ethanol, and (C) ThinPrep liquid-based preparation. Protocols C and A were also evaluated using real FNABs. Results show that protocol C markedly decreased the amount of haemoglobin, with respect to protocols A and B. Protein profiles obtained with protocols A and B were characterised by high inter-patient variability, probably related to the abundance of the haemoglobin, whereas similar spectra were observed for protocol C, where haemoglobin contents were lower. Our findings suggest protocol C as the sample preparation method for MALDI-MSI analysis. Graphical abstract.

MALDI-MSI of Immunotherapy: Mapping the EGFR-Targeting Antibody Cetuximab in 3D Colon-Cancer Cell Cultures.

Immunotherapies are treatments that use a patient's immune system to combat disease. One important type of immunotherapy employed in cancer treatments is the delivery of monoclonal antibodies to block growth receptors. In this manuscript, we develop a methodology that enables accurate and simple evaluation of antibody-type drug delivery using MALDI-MSI. To overcome the mass-range limitation that prevents the detection of large therapeutic antibodies, we used in situ reduction and alkylation to break disulfide bonds to generate smaller fragments. These smaller fragments are more readily ionized and detected by MALDI-MSI without loss of spatial information on the parent drug. As a proof of concept study, we evaluated the distribution of cetuximab in 3D colon cell cultures. Cetuximab is a monoclonal antibody that binds to the extracellular domain of epidermal-growth-factor receptor (EGFR), which is often overexpressed in colorectal cancer (CRC) and mediates cell differentiation, proliferation, migration, and angiogenesis. Cetuximab directly inhibits tumor growth and metastasis and induces apoptosis. By performing on-tissue reduction followed by MALDI-MSI analysis, we successfully mapped the time-dependent penetration and distribution of cetuximab in spheroids derived from two different colon-cancer cell lines (HT-29 and DLD-1). The localization patterns were further confirmed with IF staining of the drug. Changes in other biomolecules following drug treatment were also observed, including the elevation of ATP in spheroids. The developed method has also been applied to map cetuximab distribution in patient-derived colorectal-tumor organoids (CTOs). Overall, we believe this powerful label-free approach will be useful for visualizing the heterogeneous distribution of antibody drugs in tissues and tumors and will help to monitor and optimize their use in the clinic.

MALDI-MSI Pilot Study Highlights Glomerular Deposits of Macrophage Migration Inhibitory Factor as a Possible Indicator of Response to Therapy in Membranous Nephropathy.

Membranous nephropathy (MN) is the most frequent cause of nephrotic syndrome in adults and the disease course is characterized by the "rule of third", with one-third of patients experiencing complete remission and the remaining experiencing relapses or progression of the disease. Additionally, the therapeutic approach is not standardized, leading to further heterogeneity in terms of response and outcome. In this pilot study, MALDI-MSI analysis is performed on renal biopsies (n = 13) obtained from two homogeneous groups of patients, which differentially responded to the immunosuppressive treatments (Ponticelli regimen). A signal at m/z 1303 displays the greatest discriminatory power when comparing the two groups and is observed to be of higher intensity in the glomeruli of the non-responding patients. The corresponding tryptic peptide is identified as macrophage migration inhibitory factor (MIF). Despite much effort being made in recent years to understand the pathogenesis of MN, a biomarker able to predict the outcome of these patients following therapeutic treatment is still lacking. Here, a protein (MIF), verified by immunohistochemistry, that can differentiate between these MN patients and could be a valuable starting point for a further study focused on verifying its predictive role in therapy response is highlighted.

Digital PCR After MALDI-Mass Spectrometry Imaging to Combine Proteomic Mapping and Identification of Activating Mutations in Pulmonary Adenocarcinoma.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) is a powerful tool to analyze the spatial distribution of peptides in tissues. Digital PCR (dPCR) is a method to reliably detect genetic mutations. Biopsy material is often limited due to minimally invasive techniques, but information on diagnosis, prognosis, and prediction is required for subsequent clinical decision making. Thus, saving tissue material during diagnostic workup is highly warranted for best patient care. The possibility to combine proteomic analysis by MALDI-MSI and mutational analysis by dPCR from the same tissue section is evaluated. Ten 0.5×0.5cm formalin-fixed paraffin embedded tissue samples of pulmonary adenocarcinomas with known EGFR or KRAS mutations are analyzed by MALDI-MSI. Subsequently, DNA is extracted from the analyzed tissue material and tested for the respective driver mutation by dPCR. Detection of driver gene mutations after MALDI MSI analysis is successful in all analyzed samples. Determined mutant allele frequencies are in good agreement with values assessed from untreated serial tissue sections with a mean absolute deviation of 0.16. It has been demonstrated that MALDI-MSI can be combined with genetic analysis, like dPCR. Workflows enabling the subsequent analysis of proteomic and genetic markers are particularly promising for the analysis of limited sample material such as biopsy specimen.

MALDI-MSI analysis revealed an increment of lipid candidate biomarkers in oa synovium

MALDI-MSI analysis revealed an increment of lipid candidate biomarkers in oa synovium

Organic washes of tissue sections for comprehensive analysis of small molecule metabolites by MALDI MS imaging of rat brain following status epilepticus.

In-situ detection and in particular comprehensive analysis of small molecule metabolites (SMMs, m/z < 500) using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) remain a challenge, mainly due to ion suppression effects from more abundant molecules in tissue section like lipids. A strategy based on organic washes to remove most ionization-suppressing lipids from tissue section was firstly explored for improved analysis of SMMs by MALDI MSI. The tissue sections after rinse with different organic solvents were analyzed by MALDI MSI, and the results were compared for the optimized washing conditions. The rinse with chloroform for 15s at - 20°C significantly removed most glycerophospholipids and glycerolipids from tissue section. Consequentially, ATP-related energy metabolites, amino acids and derivatives, glucose derivatives, glycolysis pathway metabolites and other SMMs were able to be well-visualized with enhanced ion intensity and good reproducibility. The organic washes-based MALDI MSI was applied to the metabolic pathway analysis in rat brain following status epilepticus (SE) model, which was, as far as we know, the first report about in-situ detection of a broad range of metabolites in the model of SE by MALDI MSI technique. The alterations of cyclic adenosine monophosphate (cyclic AMP), inosine, glutamine, glutathione, taurine and spermine during SE were observed. A simple organic washing protocol enables comprehensive analysis of tissue SMMs in MALDI MSI by removing ionization-suppressing lipids. The application in the SE model indicates that MALDI MSI analysis potentially provides new insight for understanding the disease mechanism.

The effect of antigen retrieval buffers on MALDI mass spectrometry imaging of peptide profiles in skin FFPE tissue

Abstract Objectives: The success of novel in-situ immunohistoproteomics approaches that employ Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry imaging (MSI) heavily depends on good antigen retrieval efficacy. Methods: We have optimized the effect of various antigen retrieval buffers on peptide profiles acquired from FFPE traction alopecia scalp tissue. Heat-induced epitope retrieval (HIER) was performed using Citrate, Tris, and Tris-EDTA buffers under various conditions. MALDI-MSI analysis of the tissue was performed using a high-throughput, state-of-the-art RapiFlex MALDI TOF/TOF tissue-typer equipped with a smart beam laser. Results: Background matrix peaks were found under most conditions except under Tris-EDTA buffer. Trypsin digestion periods longer than overnight and possibly higher concentrations are needed for Citrate and Tris. The Tris-EDTA buffer performed best for MALDI-MSI antigen retrieval at an incubation period of overnight at 60°C. Conclusions: We have identified in this study that antigen retrieval conditions need to be optimised prior to MALDI MSI analysis of different tissue types.

Silver nanoparticles as matrix for MALDI FTICR MS profiling and imaging of diverse lipids in brain

Owing to the diversity of lipids, profiling and imaging multiple classes of lipids in one analysis by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is a great challenge. In this work, polyvinylpyrrolidone (PVP) capped silver nanoparticles (AgNPs) was used as the matrix for MALDI MSI for the first time to simultaneously analyze 10 classes of lipids from the brain. This analysis included fatty acids and their derivatives, sterols, CPAs, LPA and PAs, LPE and PEs, LPC and PCs, PS, Cers, SMs, and MAGs and DAGs, and other small metabolites. Owing to the abundant silver ions on the surface of PVP-capped AgNPs, compounds with poor ionization efficiency such as FAs and sterols can be detected. The PVP-capped AgNPs based MALDI MSI analysis of mouse brain showed that lipid distributions in the substructures of the mouse brain can be connected with their biological functions. The brain lipids in rats with middle cerebral artery occlusion (MCAO) were also investigated. Most unsaturated FAs, prostaglandins, CPAs, vitamin A, neuraminic acid, 5-OH-tryptophan and the K+ adducts of most phospholipids (PAs, LPE, PEs, PCs, PS) and SMs were extremely down regulated in the ischemic region and saturated FA, Cers, hexanoylcarnitine, stearaldehyde, the Na+ adduct of phospholipids (LPA, PAs, LPE, PEs, LPC, PCs) and SMs were highly expressed in the damaged section. These novel findings could be very significant for elucidating the disease mechanism. MALDI MSI using PVP-capped AgNPs as a matrix can be a powerful tool in histopathology and pathology studies.

MALDI MS imaging investigation of the host response to visceral leishmaniasis.

Mass spectrometry imaging (MSI) of animal tissues has become an important tool for in situ molecular analyses and biomarker studies in several clinical areas, but there are few applications in parasitological studies. Leishmaniasis is a neglected tropical disease, and experimental mouse models have been essential to evaluate pathological and immunological processes and to develop diagnostic methods. Herein we have employed MALDI MSI to examine peptides and low molecular weight proteins (2 to 20 kDa) differentially expressed in the liver during visceral leishmaniasis in mice models. We analyzed liver sections of Balb/c mice infected with Leishmania infantum using the SCiLS Lab software for statistical analysis, which facilitated data interpretation and thus highlighted several key proteins and/or peptides. We proposed a decision tree classification for visceral leishmaniasis with distinct phases of the disease, which are named here as healthy, acute infection and chronic infection. Among others, the ion of m/z 4963 was the most important to identify acute infection and was tentatively identified as Thymosin β4. This peptide was previously established as a recovery factor in the human liver and might participate in the response of mice to Leishmania infection. This preliminary investigation shows the potential of MALDI MSI to complement classical compound selective imaging techniques and to explore new features not yet recognized by these approaches.