Matrix-assisted Laser Desorption/ionization Mass Spectrometry Imaging Analysis Research Articles

Novel Small Molecule Matrix Screening for Simultaneous MALDI Mass Spectrometry Imaging of Multiple Lipids and Phytohormones.

Most of the traditional matrices cannot simultaneously image multiple lipids and phytohormones, so screening and discovery of novel matrices stand as essential approaches for broadening the application scope of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). In this work, 12 organic small molecule compounds were comprehensively screened and investigated as potential MALDI matrices for simultaneous imaging analysis of various lipids and phytohormones. In the positive ionization mode, p-nitroaniline, m-nitroaniline, and 2-aminoterephthalic acid displayed good performance for the highly sensitive detection of lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), and triacylglycerols (TGs). Furthermore, p-nitroaniline possessed excellent characteristics of strong ultraviolet absorption and homogeneous cocrystallization, making it a desirable matrix for MALDI-MSI analysis of eight plant hormones. Compared with conventional matrices (2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 9-aminoacridine (9-AA), the use of p-nitroaniline resulted in higher ionization efficiency, superior sensitivity, and clearer imaging images in dual polarity mode. Our research offers valuable guidance and new ideas for future endeavors in matrix screening.

Improving the Signal Intensity of Cryosections Using a Conductive Adhesive Film in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 μm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

A sensitive on-tissue chemical derivatization-mass spectrometry imaging method for the quantitative visualization of helicid in mice

Helicid (4-formylphenyl-O-β-d-allopyranoside) is an aromatic phenolic glucoside. It is one of the main active constituents from the seeds of the Chinese herb Helicia nilagirica. Helicid has been reported to exert sedative, analgesic, hypnotic and antidepressant effects, and is currently used clinically to treat neurasthenic syndrome, vascular headaches and trigeminal neuralgia. On-tissue chemical derivatization (OTCD) combined with matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was developed for the quantitative visualization of helicid in mice. The derivatization reagent, Girard’s reagent T (GT), was sprayed directly onto the tissue surface using an automated pneumatic TM-sprayer. The OTCD reaction was conducted in a closed container with 120 mL of 50% methanol with 0.4% TFA at 40 °C for 40 min. α-Cyano-4-hydroxycinnamic acid (2.22 μg/mm2) was used as the matrix, which was deposited onto the tissue surface. MALDI-MSI analysis was performed in the positive-ion mode with a spatial resolution of 200 μm. The sensitivity for the MALDI-MSI of helicid in tissue sections increased 1920 times after derivatization. The assay showed good linearity in the helicid concentration range of 0.32–32 ng/mm2. The method was used to investigate the spatiotemporal distribution of helicid in mice.

Tracking Spatial Distribution Alterations of Multiple Endogenous Molecules during Lentil Germination by MALDI Mass Spectrometry Imaging

Exploring the spatial distribution alterations of metabolites during lentil germination is essential to reveal the nutritional value, physiological function, and metabolic pathway in lentils. Hence, an effective matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) method was established for the first time to visualize the spatial localization changes of 53 metabolites in lentils during germination for 12-72 h. The results of MALDI-MSI analysis showed that phosphatidylinositols, phosphatidylethanolamines, phosphatidylglycerols, and phosphatidic acids were mainly located in the cotyledons of lentils throughout the germination process, while triacylglycerols, phosphatidylcholines, diacylglycerols, amino acids, choline, and spermine spread throughout the lentil tissue at the initial stage of germination and gradually presented obvious distribution characteristics in the radicle with increasing germination time. Heat map analysis was used to visualize the correlations between lipid content changes and germination time, which supported the use of germinated lentils as nutraceutical or functional food.

Simultaneous metabolite MALDI-MSI, whole exome and transcriptome analysis from formalin-fixed paraffin-embedded tissue sections

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows spatial analysis of proteins, metabolites, or small molecules from tissue sections. Here, we present the simultaneous generation and analysis of MALDI-MSI, whole-exome sequencing (WES), and RNA-sequencing data from the same formalin-fixed paraffin-embedded (FFPE) tissue sections. Genomic DNA and total RNA were extracted from (i) untreated, (ii) hematoxylin-eosin (HE) stained, and (iii) MALDI-MSI-analyzed FFPE tissue sections from three head and neck squamous cell carcinomas. MALDI-MSI data were generated by a time-of-flight analyzer prior to preprocessing and visualization. WES data were generated using a low-input protocol followed by detection of single-nucleotide variants (SNVs), tumor mutational burden, and mutational signatures. The transcriptome was determined using 3'-RNA sequencing and was examined for similarities and differences between processing stages. All data met the commonly accepted quality criteria. Besides SNVs commonly identified between differently processed tissues, FFPE-typical artifactual variants were detected. Tumor mutational burden was in the same range for tissues from the same patient and mutational signatures were highly overlapping. Transcriptome profiles showed high levels of correlation. Our data demonstrate that simultaneous molecular profiling of MALDI-MSI-processed FFPE tissue sections at the transcriptome and exome levels is feasible and reliable.The authors present a workflow that allows the simultaneous measurement of the whole exome and the transcriptome by next-generation sequencing from formalin-fixed paraffin-embedded tissue sections that were analyzed by matrix-assisted laser desorption ionization mass spectrometry imaging. The data and analyses demonstrate the feasibility and reproducibility of this approach, which expands the possibilities of multi-omics integration in cancer research.

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.

Spatial distribution characteristics of metabolities in rhizome of Paris polyphylla var. yunnanensis: based on MALDI-MSI

In this study, a method was established for in-situ visualization of metabolite distribution in the rhizome of Paris polyphylla var. yunnanensis. To be specific, through matrix-assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI), the spatial locations of steroidal saponins, amino acids, organic acids, phytosterols, phytoecdysones, nucleosides, and esters in rhizome of the medicinal plant were directly analyzed, and six unknown compounds with differential distribution in rhizome tissues were identified. The specific procedure is as follows: preparation of rhizome tissue section, matrix screening and optimization, and MALDI-MSI analysis. The results showed that the steroidal saponins were mainly distributed in the central, amino acids in epidermis and cortex, low-molecular-weight organic acids in central epidermis, phytosterols in the epidermis and lateral cortex, the phytoecdysones in epidermis and cortex, nucleosides(uneven distribution) in epidermis and cortex, growth hormones around the epidermis and cortex, particularly outside the cortex, and esters in cortex with unobvious difference among different tissues. In this study, the spatial distribution of meta-bolites in the rhizome of P. polyphylla var. yunnanensis was characterized for the first time. The result can serve as a reference for identifying and extracting endogenous metabolites of P. polyphylla var. yunnanensis, exploring the synthesis and metabolism mechanisms of the metabolites, and evaluating the quality of medicinal materials.

Spatial proteomic alterations detected via MALDI-MS imaging implicate neuronal loss in a Huntington's disease mouse (YAC128) brain.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that occurs with the increase of CAG trinucleotide repeats in the huntingtin gene. To understand the mechanisms of HD, powerful proteomics techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed. However, one major drawback of these methods is loss of the region-specific quantitative information of the proteins due to analysis of total tissue lysates. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a MS-based label-free technique that works directly on tissue sections and gathers m/z values with their respective regional information. In this study, we established a data processing protocol that includes several software programs and methods to determine spatial protein alterations between the brain samples of a 12 month-old YAC128 HD mouse model and their non-transgenic littermates. 22 differentially expressed proteins were revealed with their respective regional information, and possible relationships of several proteins were discussed. As a validation of the MALDI-MSI analysis, a differentially expressed protein (GFAP) was verified using immunohistochemical staining. Furthermore, since several proteins detected in this study have previously been associated with neuronal loss, neuronal loss in the cortical region was demonstrated using an anti-NeuN immunohistochemical staining method. In conclusion, the findings of this research have provided insights into the spatial proteomic changes between HD transgenic and non-transgenic littermates and therefore, we suggest that MALDI-MSI is a powerful technique to determine spatial proteomic alterations between biological samples, and the data processing that we present here can be employed as a complementary tool for the data analysis.

Protein hyper‐glycosylation is a metabolic feature of mouse and human brains with beta amyloid pathology

Glycomics is the study of complex carbohydrates throughout their metabolic lifecycle; precise profiling of these oligosaccharides has been shown to provide a critical understanding of the current state of a disease. N-linked glycans are a subset of the glycome containing co-translational modifications found on cell surface, secreted, and circulating proteins. Prior research shows glycosylation perturbations in Alzheimer's disease (AD), however, these patterns have not been extensively studied in specific brain regions with underlying disease pathology. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a revolutionary technique that combines traditional mass spectrometry with high-resolution imaging to visualize microenvironmental glycan distribution. Using our improved, enzyme-assisted workflow, we can define sub-regional N-linked glycan features in multiple areas of the brain implicated in AD pathogenesis including the hippocampus and the frontal cortex. We saw a profound hyper N-linked glycosylation pattern in multiple (n=4) 5xFAD mouse model (beta-amyloid pathology) brain regions compared to age- and sex-matched, heterozygous littermate controls. Consistent with our in vivo findings, MALDI-MSI analysis of human AD hippocampus and frontal cortex tissue samples revealed a similar hyper glycosylation phenotype. Our studies suggest that beta-amyloid is a metabolic modifier of N-linked glycosylation and could be an exciting therapeutic approach as well as a potential biomarker for AD progression. In the future, we plan to use these results to further investigate specific glycosylation pathways that promote AD pathogenesis, and how these modifications contribute to ER stress and the unfolded protein response, inflammation, and neuronal cell death.

In Situ Localization of Plant Lipid Metabolites by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI).

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a major analytical platform for the determination and localization of lipid metabolites directly from tissue sections. Unlike analysis of lipid extracts, where lipid localizations are lost due to homogenization and/ or solvent extraction, MALDI-MSI analysis is capable of revealing spatial localization of metabolites while simultaneously collecting high chemical resolution mass spectra. Important considerations for obtaining high quality MALDI-MS images include tissue preservation, section preparation, MS data collection and data processing. Errors in any of these steps can lead to poor quality metabolite images and increases the chance for metabolite misidentification and/ or incorrect localization. Here, we present detailed methods and recommendations for specimen preparation, MALDI-MS instrument parameters, software analysis platforms for data processing, and practical considerations for each of these steps to ensure acquisition of high-quality chemical and spatial resolution data for reconstructing MALDI-MS images of plant tissues.

Spatial Analysis of Phosphatidylinositol Molecular Species in Pork Chop Tissues Using Matrix-assisted Laser Desorption/ionization-Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing lipids in biological tissues. Phosphatidylinositol (PI), a phospholipid in pork, is a major source of inositol in animal-derived foods believed to be protective against diseases related to pregnancy and cancer. However, the distribution of PI molecular species in pork is not well understood. Here, we performed MALDI-MSI analysis to investigate the distribution and composition of PI molecular species in pork chop comprising Longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Twelve diacyl-PI molecular species were identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) and MALDI-MS/MS analysis and visualized using MALDI-MSI. Spinalis muscle had the highest amount of identified PI molecular species, followed by loin, transparent tissue, and intermuscular fat tissue. The diacyl-PI molecular species containing hexadecadienoic, oleic, linoleic and eicosadienoic acids at the sn-2 position were mainly abundant in the loin and spinalis muscle, whereas those containing mead, arachidonic, docosatetraenoic, and docosapentaenoic acids at the sn-2 position were mainly abundant in both muscles as well as transparent tissues. Notably, the balance of PI molecular species differed among the tissues depending on fatty acid compositions at the sn-2 position. These results suggested that MALDI-MSI is a promising tool for assessing the association between individual pork tissues and the protective effects of PI molecular species against diseases related to pregnancy and cancer. To the best of our knowledge, this is the first report showing tissue-specific distributions of PI molecular species in pork chop using MALDI-MSI.

Abstract PO-094: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N- glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexanda E. Stanback, Derek B Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-094.

Abstract PO-085: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N-glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexandra E. Stanback, Derek B. Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-085.

Mass spectrometry imaging of latent fingerprints using titanium oxide development powder as an existing matrix.

Recent research has focused on increasing the evidentiary value of latent fingerprints through chemical analysis. Although researchers have optimized the use of organic and metal matrices for matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of latent fingerprints, the use of development powders as matrices has not been fully investigated. Carbon forensic powder (CFP), a common nonporous development technique, was shown to be an efficient one-step matrix; however, a high-resolution mass spectrometer was required in the low mass range due to carbon clusters. Titanium oxide (TiO2 ) is another commonly used development powder, especially for dark nonporous surfaces. Here, forensic TiO2 powder is utilized as a single-step development and matrix technique for chemical imaging of latent fingerprints without the requirement of a high-resolution mass spectrometer. All studied compounds were successfully detected when TiO2 was used as the matrix in positive mode, although, generally, the overall ion signals were lower than the previously studied CFP. TiO2 provided quality mass spectrometry (MS) images of endogenous and exogenous latent fingerprint compounds. The subsequent addition of traditional matrices on top of the TiO2 powder was ineffective for universal detection of latent fingerprint compounds. Forensic TiO2 development powder works as an efficient single-step development and matrix technique for MALDI-MSI analysis of latent fingerprints in positive mode and does not require a high-resolution mass spectrometer for analysis.

Conductive Adhesive Film Expands the Utility of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique is a promising approach for detecting the distribution of small molecules in a section of biological tissue. However, when a cryosection is created from fragile, hard, or whole-body samples, obtaining a high-quality section that maintains the distribution of the various components has been difficult. Since adhesive films have the potential to obtain high-quality cryosections, we attempted to utilize a conductive adhesive film for MALDI-MSI. To this end, cryosections of the whole body of a 9-day-old mouse were directly prepared on indium tin oxide (ITO) glass slides, nonconductive adhesive films, or conductive adhesive films, and the signal intensities from each section were measured by MALDI-MSI. We measured the differences in the ion intensity among these three slides/films by means of multivariate analyses and found that both the nonconductive and conductive adhesive films gave rise to high-quality sections in comparison with the ITO glass slide. The conductive adhesive film gave higher signals that were comparable to those of the ITO glass slide in comparison with the nonconductive adhesive film. We divided the frozen sections into two groups, a freeze-dried group and a thawed group, to examine the freeze-thaw effect on the signals of representative compounds of amino acids, cholesterol, and phosphatidylcholines. The freeze-dried samples were found to be useful for the analysis. These results indicate that the sections made with the conductive adhesive film under a freeze-dried condition can expand the utility of the MALDI-MSI analysis.

High Spatial Resolution MALDI-MS Imaging in the Study of Membranous Nephropathy.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technology has advanced rapidly during recent years with the development of instruments equipped with low-diameter lasers that are suitable for high spatial resolution imaging. This may provide significant advantages in certain fields of molecular pathology where more specific protein fingerprints of individual cell types are required, such as renal pathology. Here MALDI-MSI analysis of a cohort of membranous nephropathy (MN) patients is performed among which patients either responded favorably (R; n=6), or unfavorably (NR; n=4), to immunosuppressive treatment (Ponticelli Regimen), employing a 10µm laser spot diameter. Specific tryptic peptide profiles of the different cellular regions within the glomerulus can be generated, similarly for the epithelial cells belonging to the proximal and distal tubules. Conversely, specific glomerular and sub-glomerular profiles cannot be obtained while using the pixel size performed in previous studies (50µm). Furthermore, two proteins are highlighted, sonic hedgehog and α-smooth muscle actin, whose signal intensity and spatial localization within the sub-glomerular and tubulointerstitial compartments differ between treatment responders and non-responders. The present study exemplifies the advantage of using high spatial resolution MALDI-MSI for the study of MN and highlights that such findings have the potential to provide complementary support in the routine prognostic assessment of MN patients.

Combination of ESI and MALDI mass spectrometry for qualitative, semi-quantitative and in situ analysis of gangliosides in brain.

Gangliosides are a family of complex lipids that are abundant in the brain. There is no doubt the investigations about the distribution of gangliosides in brian and the relationship between gangliosides and Alzheimer’s disease is profound. However, these investigations are full of challenges due to the structural complexity of gangliosides. In this work, the method for efficient extraction and enrichment of gangliosides from brain was established. Moreover, the distribution of gangliosides in brain was obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). It was found that 3-aminoquinoline (3-AQ) as matrix was well-suited for MALDI MS analysis of gangliosides in negative ion mode. In addition, the pretreatment by ethanol (EtOH) cleaning brain section and the addition of ammonium formate greatly improved the MS signal of gangliosides in the brain section when MALDI MSI analysis was employed. The distribution of ganliosides in cerebral cortex, hippocampus and cerebellum was respectively acquired by electrospray ionization (ESI) MS and MALDI MSI, and the data were compared for reliability evaluation of MALDI MSI. Further, applying MALDI MSI technology, the distribution of gangliosides in amyloid precursor protein transgenic mouse brain was obtained, which may provide a new insight for bioresearch of Alzheimer’s disease (AD).

Investigating MALDI MSI parameters (Part 2) – On the use of a mechanically shuttered trigger system for improved laser energy stability

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is now widely used to desorb, ionize and detect molecules from complex samples and tissue sections. The detected ion intensity within MALDI MS and MSI is intimately linked to the laser energy per pulse incident upon the sample during analysis. Laser energy/power stability can be significantly affected by the manner in which the laser is operated. High-repetition rate diode-pumped solid-state (DPSS) lasers are being increasingly adopted to enable high-throughput MALDI MSI analysis. Within this work two different laser-triggering setups are used to demonstrate the effect of laser energy instabilities due to spiking and thermal control phenomena and a setup with a shutter to remove these effects. The effect of non-equilibrium laser operation on MALDI MSI data versus the more stable laser pulse energy of the shutter-triggered system is demonstrated in thin films of α-cyano-4-hydroxycinnamic acid (CHCA) and for imaging of murine brain tissue sections. Significant unwanted variations in absolute and relative detected ion intensity are shown where energy variation is introduced by these phenomena, which return to equilibrium within the setup employed here over timescales relevant to MALDI MS analysis.

Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator

AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway.

MALDI mass spectrometry imaging analysis of pituitary adenomas for near-real-time tumor delineation

We present a proof of concept study designed to support the clinical development of mass spectrometry imaging (MSI) for the detection of pituitary tumors during surgery. We analyzed by matrix-assisted laser desorption/ionization (MALDI) MSI six nonpathological (NP) human pituitary glands and 45 hormone secreting and nonsecreting (NS) human pituitary adenomas. We show that the distribution of pituitary hormones such as prolactin (PRL), growth hormone (GH), adrenocorticotropic hormone (ACTH), and thyroid stimulating hormone (TSH) in both normal and tumor tissues can be assessed by using this approach. The presence of most of the pituitary hormones was confirmed by using MS/MS and pseudo-MS/MS methods, and subtyping of pituitary adenomas was performed by using principal component analysis (PCA) and support vector machine (SVM). Our proof of concept study demonstrates that MALDI MSI could be used to directly detect excessive hormonal production from functional pituitary adenomas and generally classify pituitary adenomas by using statistical and machine learning analyses. The tissue characterization can be completed in fewer than 30 min and could therefore be applied for the near-real-time detection and delineation of pituitary tumors for intraoperative surgical decision-making.