MALDI Imaging Research Articles

Adolescent Δ-9-tetrahydrocannabinol exposure induces differential acute and long-term neuronal and molecular disturbances in dorsal vs. ventral hippocampal subregions.

Chronic exposure to Δ-9-tetrahydrocannabinol (THC) during adolescence is associated with long-lasting cognitive impairments and enhanced susceptibility to anxiety and mood disorders. Previous evidence has revealed functional and anatomical dissociations between the posterior vs. anterior portions of the hippocampal formation, which are classified as the dorsal and ventral regions in rodents, respectively. Notably, the dorsal hippocampus is critical for cognitive and contextual processing, whereas the ventral region is critical for affective and emotional processing. While adolescent THC exposure can induce significant morphological disturbances and glutamatergic signaling abnormalities in the hippocampus, it is not currently understood how the dorsal vs. ventral hippocampal regions are affected by THC during neurodevelopment. In the present study, we used an integrative combination of behavioral, molecular, and neural assays in a neurodevelopmental rodent model of adolescent THC exposure. We report that adolescent THC exposure induces long-lasting memory deficits and anxiety like-behaviors concomitant with a wide range of differential molecular and neuronal abnormalities in dorsal vs. ventral hippocampal regions. In addition, using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), we show for the first time that adolescent THC exposure induces significant and enduring dysregulation of GABA and glutamate levels in dorsal vs. ventral hippocampus. Finally, adolescent THC exposure induced dissociable dysregulations of hippocampal glutamatergic signaling, characterized by differential glutamatergic receptor expression markers, profound alterations in pyramidal neuronal activity and associated oscillatory patterns in dorsal vs. ventral hippocampal subregions.

Protein Alterations in Cardiac Ischemia/Reperfusion Revealed by Spatial-Omics.

Myocardial infarction is the most common cause of death worldwide. An understanding of the alterations in protein pathways is needed in order to develop strategies that minimize myocardial damage. To identify the protein signature of cardiac ischemia/reperfusion (I/R) injury in rats, we combined, for the first time, protein matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and label-free proteomics on the same tissue section placed on a conductive slide. Wistar rats were subjected to I/R surgery and sacrificed after 24 h. Protein MALDI-MSI data revealed ischemia specific regions, and distinct profiles for the infarct core and border. Firstly, the infarct core, compared to histologically unaffected tissue, showed a significant downregulation of cardiac biomarkers, while an upregulation was seen for coagulation and immune response proteins. Interestingly, within the infarct tissue, alterations in the cytoskeleton reorganization and inflammation were found. This work demonstrates that a single tissue section can be used for protein-based spatial-omics, combining MALDI-MSI and label-free proteomics. Our workflow offers a new methodology to investigate the mechanisms of cardiac I/R injury at the protein level for new strategies to minimize damage after MI.

Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging.

Spatial metabolomics describes the spatially resolved analysis of interconnected pathways, biochemical reactions, and transport processes of small molecules in the spatial context of tissues and cells. However, a broad range of metabolite classes (e.g., steroids) show low intrinsic ionization efficiencies in mass spectrometry imaging (MSI) experiments, thus restricting the spatial characterization of metabolic networks. Additionally, decomposing complex metabolite networks into chemical compound classes and molecular annotations remains a major bottleneck due to the absence of repository-scaled databases. Here, we describe a multimodal mass-spectrometry-based method combining computational metabolome mining tools and high-resolution on-tissue chemical derivatization (OTCD) MSI for the spatially resolved analysis of metabolic networks at the low micrometer scale. Applied to plant toxin sequestration in Danaus plexippus as a model system, we first utilized liquid chromatography (LC)-MS-based molecular networking in combination with artificial intelligence (AI)-driven chemical characterization to facilitate the structural elucidation and molecular identification of 32 different steroidal glycosides for the host-plant Asclepias curassavica. These comprehensive metabolite annotations guided the subsequent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) analysis of cardiac-glycoside sequestration in D. plexippus. We developed a spatial-context-preserving OTCD protocol, which improved cardiac glycoside ion yields by at least 1 order of magnitude compared to results with untreated samples. To illustrate the potential of this method, we visualized previously inaccessible (sub)cellular distributions (2 and 5 μm pixel size) of steroidal glycosides in D. plexippus, thereby providing a novel insight into the sequestration of toxic metabolites and guiding future metabolomics research of other complex sample systems.

Quantitative Analyses and Validation of Phospholipids and Sphingolipids in Ischemic Rat Brains.

Prior MALDI mass spectrometry imaging (MALDI-MSI) studies reported significant changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), and sphingomyelins (SMs) in ischemic rat brains yet overlooked the information on other classes of PLs and SLs and provided very little or no validation on the detected lipid markers. Relative quantitation of four classes of PLs and two classes of SLs in the ischemic and normal temporal cortex (TCX), parietal cortex (PCX), and striatum (ST) of rats was performed with hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS/MS) analyses, and the marker lipid species was identified by multivariate data analysis and validated with additional tissue cohorts. The acquired lipid information was sufficient in differentiating individual anatomical regions under different pathological states, identifying region-specific ischemic brain lipid markers and revealing additional PL and SL markers not reported previously. Validation of orthogonal partial least square discriminating analysis (OPLS-DA) identified ischemic brain lipid markers yielded much higher classification accuracy, precision, specificity, sensitivity, and lower false positive and false negative rates than those from the volcano plot analyses using conventional statistical significance and a fold change of two as the cutoff and provided a wider prospective to ischemia-associated brain lipid changes.

Boron Delivery to Brain Cells via Cerebrospinal Fluid (CSF) Circulation in BNCT of Brain-Tumor-Model Rats-Ex Vivo Imaging of BPA Using MALDI Mass Spectrometry Imaging.

The blood-brain barrier (BBB) is likely to be intact during the early stages of brain metastatic melanoma development, and thereby inhibits sufficient drug delivery into the metastatic lesions. Our laboratory has been developing a system for boron drug delivery to brain cells via cerebrospinal fluid (CSF) as a viable pathway to circumvent the BBB in boron neutron capture therapy (BNCT). BNCT is a cell-selective cancer treatment based on the use of boron-containing drugs and neutron irradiation. Selective tumor targeting by boron with minimal normal tissue toxicity is required for effective BNCT. Boronophenylalanine (BPA) is widely used as a boron drug for BNCT. In our previous study, we demonstrated that application of the CSF administration method results in high BPA accumulation in the brain tumor even with a low dose of BPA. In this study, we evaluate BPA biodistribution in the brain following application of the CSF method in brain-tumor-model rats (melanoma) utilizing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). We observed increased BPA penetration to the tumor tissue, where the color contrast on mass images indicates the border of BPA accumulation between tumor and normal cells. Our approach could be useful as drug delivery to different types of brain tumor, including brain metastases of melanoma.

Pyrylium-Based Derivatization for Rapid Labeling and Enhanced Detection of Cholesterol in Mass Spectrometry Imaging.

Cholesterol in the central nervous system has been increasingly found to be closely related to neurodegenerative diseases. Defects in cholesterol metabolism can cause structural and functional disorders of the central nervous system. The detection of abnormal cholesterol is of great significance for the cognition of physiological and pathological states of organisms, and the spatial distribution of cholesterol can also provide more clues for our understanding of the complex mechanism of disease. Here, we developed a novel pyrylium-based derivatization reagent combined with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to visualize cholesterol in biological tissues. A new class of charged hydroxyl derivatization reagents was designed and synthesized, and finally 1-(carboxymethyl)-2,4,6-trimethylpyridinium (CTMP) was screened for tissue derivatization of cholesterol. Different from the shortcomings of traditional hydroxyl labeling methods such as harsh reaction conditions and long reaction time, in our study, we combined the advantages of CTMP itself and the EDCl/HOBt reaction system to achieve instant labeling of cholesterol on tissues through two-step activation. In addition, we also reported changes in cholesterol content in different stages and different brain regions during disease development in SOD1 mutant mouse model. The cholesterol derivatization method we developed provides an efficient way to explore the distribution and spatial metabolic network of cholesterol in biological tissues.

Device-Controlled Microcondensation for Spatially Confined On-Tissue Digests in MALDI Imaging of N-Glycans.

On-tissue enzymatic digestion is a prerequisite for MALDI mass spectrometry imaging (MSI) and spatialomic analysis of tissue proteins and their N-glycan conjugates. Despite the more widely accepted importance of N-glycans as diagnostic and prognostic biomarkers of many diseases and their potential as pharmacodynamic markers, the crucial sample preparation step, namely on-tissue digestion with enzymes like PNGaseF, is currently mainly carried out by specialized laboratories using home-built incubation arrangements, e.g., petri dishes placed in an incubator. Standardized spatially confined enzyme digests, however, require precise control and possible regulation of humidity and temperature, as high humidity increases the risk of analyte dislocation and low humidity compromises enzyme function. Here, a digestion device that controls humidity by cyclic ventilation and heating of the slide holder and the chamber lid was designed to enable controlled micro-condensation on the slide and to stabilize and monitor the digestion process. The device presented here may help with standardization in MSI. Using sagittal mouse brain sections and xenografted human U87 glioblastoma cells in CD1 nu/nu mouse brain, a device-controlled workflow for MALDI MSI of N-glycans was developed.

Matrix-assisted laser desorption/ionization mass spectrometry imaging reveals “Spatial-Temporal-Content” changes of parishins in Gastrodiae Rhizoma during the steaming process

Gastrodiae Rhizoma is a commonly used plant material for both medicine and food in China for thousands of years. Steaming as the main pre-processing method of Gastrodiae Rhizoma is the key to its quality formation. In this study, we established a high-coverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) method to visualize the spatial distribution of phenols in Gastrodiae Rhizoma. By optimizing 1,5-diaminonaphthalene as MALDI matrix in negative mode, the “spatial-temporal-content” changes of 13 phenols including 11 parishins in Gastrodiae Rhizoma during the steaming process were imaged. We also characterized the activity of two hydrolases related to parishins degradation during the steaming. Moreover, the degradation pathways of parishins in Gastrodiae Rhizoma were summarized. The integration of spatially-resolved parishins and corresponding hydrolases information revealed the scientific connotation of steaming as the processing method of Gastrodiae Rhizoma is to inactivate hydrolases and protect parishins, which provided theoretical support for the quality improvement and the standardized processing of Gastrodiae Rhizoma.

In-situ detection and imaging of Areca catechu fruit alkaloids by MALDI-MSI

Arecoline, the predominant alkaloid in the fruit of Areca catechu, is considered the fourth most consumed addictive drug in the world, after nicotine, alcohol, and caffeine. Additionally, the areca nut (AN) is widely used in traditional and herbal medicines. Therefore, performing an exact analysis of alkaloids will provide evidence for more optimal applications of AN. Here, we performed matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and image 10 physiologically active alkaloids in situ (arecoline, arecaidine, caffeine, cotinine, guvacine, guvacoline, hordenine, sophoridine, trigonelline, and vicine) in areca fruits at three different developmental stages. The ion images obtained by MALDI-MSI describe in detail the spatial distributions in different tissues of the fresh fruit of A. catechu, i.e., exocarp, mesocarp, endocarp, episperm, endosperm, and embryos. Liquid chromatography coupled with tandem MS (LC-MS/MS) was also carried out to verify the relative abundance and confirm the quantitative distribution of these alkaloids in Pericarpium Arecae and Semen Arecae. In this study, 3,4-dimethoxycinnamic acid (DMCA) was evaluated as a new matrix for enhanced low-MW compound detection by MALDI-MSI in A. catechu fruit for the first time. New insights into the physiological activities and pharmaceutical properties of areca fruits for further applications can be obtained.

Lipid Profiles Obtained from MALDI Mass Spectrometric Imaging in Liver Cancer Metastasis Model

Liver cancer metastasis is known to be a poor prognosis and a leading cause of mortality. To overcome low therapeutic efficacy, understanding the physiological properties of liver cancer metastasis is required. However, the metastatic lesion is heterogeneous and complex. We investigate the distribution of lipids using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in an experimental metastasis model. We obtained the differentially expressed mass peaks in comparison between normal sites and metastatic lesions. The relationship of mass to charge ratio (m/z) and intensity were measured, m/z-indicated species were analyzed by MALDI-MS/MS analysis, and identification of these mass species was confirmed using the METASPACEannotation platform and Lipid Maps®. MALDI-MSI at m/z 725.6, 734.6, 735.6, 741.6, 742.6, 744.6, 756.6, and 772.6 showed significantly higher intensity, consistent with the metastatic lesions in hematoxylin-stained tissues. Sphingomyelin SM [d18:0/16:1], phosphatidylcholine (PC) [32:0], PC [31:0], PC [31:1], and PE [36:2] were highly expressed in metastatic lesions. Our results could provide information for understanding metastatic lesions. It suggests that the found lipids could be a biomarker for the diagnosis of metastatic lesions.

Applying imaging mass spectrometry to define the N-glycan profiles of co-localized virus and immune cell infiltrates in post-COVID-19 infected lung autopsy tissues

The current COVID-19 pandemic is characterized by a broad range of disease severity in patients. This diversity in clinical manifestations has complicated our understanding of the SARS-CoV-2 pathogenesis and highlights the significance of an individual’s ability to mount an effective viral immune response. Glycosylation is a common post-translational modification occurring in complex organisms and is imperative for immune cell function. In this study, a combination approach with immunohistochemistry (IHC) and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was utilized to determine the spatial distribution of N-glycans and immune cell populations in COVID-19 lung tissues. Tissues from seven SARS-CoV-2, PCR + donors were analyzed. Tissues represented a spectrum of time spent on ventilators which was reflected in their respective viral infection status and lung pathologies. N-glycan distributions in the MALDI-IMS images were then correlated with H&E staining and IHC of SARS-CoV-2 spike protein, CD4, CD8, CD163 and CD11b. Distinct and shared N-glycan signatures were identified in association with specific immune cell types, and their co-localization with the viral spike protein. Additionally, we observed unique patterns of α2,3-linked and α2,6-linked sialic acid glycans that associated with both immune cell populations and fibrotic regions within the tissue architecture. N-glycan MALDI-IMS is an effective tool to further understand tissue-localized immune cell populations in response to emerging viral pathogens such as SARS-CoV-2.

A mass spectrometry imaging and lipidomic investigation reveals aberrant lipid metabolism in the orthotopic mouse glioma

Lipids perform multiple biological functions and reflect the physiology and pathology of cells, tissues, and organs. Here, we sought to understand lipid content in relation to tumor pathology by characterizing phospholipids and sphingolipids in the orthotopic mouse glioma using MALDI MS imaging (MSI) and LC-MS/MS. Unsupervised clustering analysis of the MALDI-MSI data segmented the coronal tumoral brain section into 10 histopathologically salient regions. Heterogeneous decrease of the common saturated phosphatidylcholines (PCs) in the tumor was accompanied by the increase of analogous PCs with one or two additional fatty acyl double bonds and increased lyso-PCs. Polyunsaturated fatty acyl-PCs and ether PCs highlighted the striatal tumor margins, whereas the distributions of other PCs differentiated the cortical and striatal tumor parenchyma. We detected a reduction of SM d18:1/18:0 and the heterogeneous mild increase of SM d18:1/16:0 in the tumor, whereas ceramides accumulated only in a small patch deep in the tumoral parenchyma. LC-MS/MS analyses of phospholipids and sphingolipids complemented the MALDI-MSI observation, providing a snapshot of these lipids in the tumor. Finally, the proposed mechanisms responsible for the tumoral lipid changes were contrasted with our interrogation of gene expression in human glioma. Together, these lipidomic results unveil the aberrant and heterogeneous lipid metabolism in mouse glioma where multiple lipid-associated signaling pathways underline the tumor features, promote the survival, growth, proliferation, and invasion of different tumor cell populations, and implicate the management strategy of a multiple-target approach for glioma and related brain malignancies.

Evaluation of Proteomic and Lipidomic Changes in Aeromonas-Infected Trout Kidney Tissue with the Use of FT-IR Spectroscopy and MALDI Mass Spectrometry Imaging.

Aeromonas species are opportunistic bacteria causing a vast spectrum of human diseases, including skin and soft tissue infections, meningitis, endocarditis, peritonitis, gastroenteritis, and finally hemorrhagic septicemia. The aim of our research was to indicate the molecular alterations in proteins and lipids profiles resulting from Aeromonas sobria and A. salmonicida subsp. salmonicida infection in trout kidney tissue samples. We successfully applied FT-IR (Fourier transform infrared) spectroscopy and MALDI-MSI (matrix-assisted laser desorption/ionization mass spectrometry imaging) to monitor changes in the structure and compositions of lipids, secondary conformation of proteins, and provide useful information concerning disease progression. Our findings indicate that the following spectral bands’ absorbance ratios (spectral biomarkers) can be used to discriminate healthy tissue from pathologically altered tissue, for example, lipids (CH2/CH3), amide I/amide II, amide I/CH2 and amide I/CH3. Spectral data obtained from 10 single measurements of each specimen indicate numerous abnormalities concerning proteins, lipids, and phospholipids induced by Aeromonas infection, suggesting significant disruption of the cell membranes. Moreover, the increase in the content of lysolipids such as lysophosphosphatidylcholine was observed. The results of this study suggest the application of both methods MALDI-MSI and FT-IR as accurate methods for profiling biomolecules and identifying biochemical changes in kidney tissue during the progression of Aeromonas infection.

N-terminal peptide fragment constitutes core of amyloid deposition of serum amyloid A: An imaging mass spectrometry study.

Serum amyloid A (SAA) is an acute phase protein, which undergoes structural changes and deposits in the extracellular matrix, causing organ damage. Systemic AA amyloidosis is a relatively common amyloid subtype among the more than 30 amyloid subtypes, but the mechanism of amyloid fibril formation remains unclear. In this study, we investigated the tissue distribution of SAA derived peptides in formalin-fixed paraffin embedded (FFPE) specimens of human myocardium with amyloidosis using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). In the whole SAA protein, four trypsin-digested peptides in the range of SAA2-67 were visualized and the N-terminal peptide; SAA2-15, was selectively localized in the Congo red-positive region. The C-terminal peptides; SAA47-62, SAA48-62, and SAA63-67 were detected not only in the Congo red-positive region but also in the surrounding negative region. Our results demonstrate that the N-terminal SAA2-15 plays a critical role in the formation of AA amyloid fibril, as previously reported. Roles of the C-terminal peptides require further investigation.

Exploring Bacterial Resistant Metabolism by Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging

The inappropriate and excessive use of antibiotics for the treatment of bacterial infections has led to the increasing presence of resistant and multidrug-resistant bacteria both in hospital settings and in the community. Thus, understanding the metabolism of resistant bacteria is extremely important to combat them more efficiently. In this scenario, mass spectrometry imaging (MSI) is considered a promising technique for understanding the resistant characteristics of such bacteria and how they can potentially be treated. This process consists of the identification of different ions on the surface of the colonies and the identification of potential metabolites that characterize antibiotic resistance, upon comparison with susceptible bacteria of the same species. This work presents matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) study of colonies of Methicillin-resistant Staphylococcus aureus, as a proof of concept of the technique for obtaining images of bacteria colonies. Images of methicillin-resistant and susceptible colonies of Staphylococcus aureus were obtained by a sublimation process to apply the MALDI matrix on the samples followed by MALDI-MSI analysis. Seventeen (17) potential metabolites were identified and spatially localized, such as N,N-dihydroxy-L-valine, 2-(4-Methylphenyl)ethylamine, 3,4-Dihydroxy-L-phenylalanine, 2-Methyl-hexanoic acid, threonine, Arginine, Aureusimine and Glycyl-D-asparagine. Thus, this study reinforces the potential of MALDI-MSI for identification of metabolites synthesized by different strains of Staphylococcus aureus bacteria.

Altered prefrontal neurochemistry in the DJ-1 knockout mouse model of Parkinson's disease: complementary semi-quantitative analyses with in vivo magnetic resonance spectroscopy and MALDI-MSI.

In vivo proton magnetic resonance spectroscopy (1H-MRS) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are two semi-quantitative analytical methods commonly used in neurochemical research. In this study, the two methods were used complementarily, in parallel, to investigate neurochemical perturbations in the medial prefrontal cortex (mPFC) of 9-month-old DJ-1 knockout mice, a well-established transgenic model for Parkinson's diseases. Convergingly, the results obtained with the two methods demonstrated that, compared with the wild-type (WT) mice, the DJ-1 knockout mice had significantly increased glutathione (GSH) level and GSH/glutamate (Glu) ratio in the mPFC, which likely presented an astrocytic compensatory mechanism in response to elevated regional oxidative stress induced by the loss of DJ-1 function. The results from this study also highlighted (1) the need to be cautious when interpreting the in vivo 1H-MRS results obtained from aged transgenic animals, in which the concentration of internal reference, being whether water or total creatine, could no longer be assumed to be the same as that in the age-matched WT animals, and (2) the necessity and importance of complementary analyses with more than one method under such circumstances.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging: Diagnostic Pathways and Metabolites for Renal Tumor Entities

Background: Correct tumor subtyping of primary renal tumors is essential for treatment decision in daily routine. Most of the tumors can be classified based on morphology alone. Nevertheless, some diagnoses are difficult, and further investigations are needed for correct tumor subtyping. Besides histochemical investigations, high-mass-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect new diagnostic biomarkers and hence improve the diagnostic. Patients and Methods: Formalin-fixed paraffin embedded tissue specimens from clear cell renal cell carcinoma (ccRCC, n = 552), papillary renal cell carcinoma (pRCC, n = 122), chromophobe renal cell carcinoma (chRCC, n = 108), and renal oncocytoma (rO, n = 71) were analyzed by high-mass-resolution MALDI fourier-transform ion cyclotron resonance (FT-ICR) MSI. The SPACiAL pipeline was executed for automated co-registration of histological and molecular features. Pathway enrichment and pathway topology analysis were performed to determine significant differences between RCC subtypes. Results: We discriminated the four histological subtypes (ccRCC, pRCC, chRCC, and rO) and established the subtype-specific pathways and metabolic profiles. rO showed an enrichment of pentose phosphate, taurine and hypotaurine, glycerophospholipid, amino sugar and nucleotide sugar, fructose and mannose, glycine, serine, and threonine pathways. ChRCC is defined by enriched pathways including the amino sugar and nucleotide sugar, fructose and mannose, glycerophospholipid, taurine and hypotaurine, glycine, serine, and threonine pathways. Pyrimidine, amino sugar and nucleotide sugar, glycerophospholipids, and glutathione pathways are enriched in ccRCC. Furthermore, we detected enriched phosphatidylinositol and glycerophospholipid pathways in pRCC. Conclusion: In summary, we performed a classification system with a mean accuracy in tumor discrimination of 85.13%. Furthermore, we detected tumor-specific biomarkers for the four most common primary renal tumors by MALDI-MSI. This method is a useful tool in differential diagnosis and biomarker detection.

Spatial-temporal lipidomics profile of acute myocardial injury

Abstract Background Lipidome disturbance has long been recognized to occur after myocardial infarction (MI). Accumulation of excessive fatty acids induces production of reactive oxygen species and consequently deteriorates cardiac injury in MI. However, the spatial and temporal lipid profile in the heart following ischemic injury remains unknown. Purpose We aim to uncover the temporal-spatial lipidome profile of the heart following ischemia reperfusion (I/R) injury and identify circulating lipids released from injured myocardium that are potentially useful for diagnosis of ischemic heart disease. Methods C57/BL6 mice were subjected to 30 min myocardial ischemia followed by removal of the ligature to establish reperfusion injury. Porcine I/R injury was induced by 105 min myocardial ischemia followed by reperfusion. Human plasma was obtained from 143 post-MI patients. Myocardial lipid profiles were generated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MALDI-MSI) in different regions (infarct, remote and peri-infarct) at different time points. Moreover, the lipids in the heart and plasma were analysed by LC-MS/MS. Results We observed a drastic alteration in the lipidome with distinct spatial-temporal features in the injured heart by both MALDI-MSI and LC-MS/MS. In the infarct heart tissue, as revealed by LC-MS/MS, we observed an elevation of glycerolipids that peaked at 3 hours after I/R, and a sustained elevation of phospholipids and sphingolipids up to 3 days. Similar alternations in lipid profile was observed but much weaker in the remote and peri-infarct heart tissue compared to the infarct tissue. Among those lipids, PC 32:0 detected by MALDI-MSI highly overlapped CD68 staining at a single-cell level, showing a strong correlation of PC 32:0 with macrophage infiltration in mouse hearts (R2=0.93, p<0.0001). A similar increase of PC 32:0 in the infarct area was also observed in porcine hearts following I/R injury. Surprisingly, plasma levels of PC 32:0 in the mice decreased after I/R injury. In humans, plasma levels of PC 32:0 in post-MI patients were lower than that in healthy individuals (p=0.03). Further analysis demonstrated that plasma levels of PC 32:0 determined within 72 hours after percutaneous coronary intervention were negatively correlated with the 6-month post-MI cardiac ejection fraction in patients (R2=0.08, p<0.001). Conclusions A temporal-spatial lipidome profile was established in heart injury by synergizing LC-MS/MS and mass spectrometry imaging. PC 32:0 levels are positively correlated with myocardial macrophage infiltration but negatively correlated with cardiac function in cardiac I/R injury. Our findings indicate that PC 32:0 is a potential biomarker for cardiac injury and the inflammatory status in the injured heart. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Singapore Ministry of Health's National Medical Research Council

Chemical traits of cerebral amyloid angiopathy in familial British-, Danish-, and non-Alzheimer's dementias.

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/β-amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aβ x-42 and Aβ x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aβ3pE-40 and Aβ3-40 but not with Aβx-42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co-aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. Cover Image for this issue: https://doi.org/10.1111/jnc.15424.

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein–protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide “molecular pictures”, which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.