Spatial flavor metabolomics: probing food flavor distribution with MALDI-MSI technology.

Mass spectrometry imaging (MSI) as a label-free and powerful imaging technique enables in situ evaluation of a tissue metabolome and/or proteome, becoming increasingly popular in the detection of plant endogenous molecules. The characterisation of structure and spatial information of endogenous molecules in plants are both very important aspects to better understand the physiological mechanism of plant organism. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a commonly-used tissue imaging technique, which requires matrix to assist in situ detection of a variety of molecules on the surface of a tissue section. In previous studies, MALDI-MSI was mostly used for the detection of molecules from animal tissue sections, compared to plant samples due to cell structural limitations, such as plant cuticles, epicuticular waxes, and cell walls. Despite the enormous progress that has been made in tissue imaging, there is still a challenge for MALDI-MSI suitable for the imaging of endogenous compounds in plants. This review summarises the recent advances in MALDI-MSI, focusing on the application of in situ detection of endogenous molecules in different plant organs, i.e. root, stem, leaf, flower, fruit, and seed. Further improvements on instrumentation sensitivity, matrix selection, image processing and sample preparation will expand the application of MALDI-MSI in plant research.

Prostate cancer is often heterogeneous with multiple tumor foci within the same prostate. MALDI (Matrix-Assisted Laser Desorption/Ionization) mass spectrometry imaging (MSI) allows for a comprehensive profiling of the molecular phenotype on the protein, peptide, lipid, and metabolite level across entire tissue slides without prior enrichment of tumor cells. This study aims in a MALDI-based discrimination of cancer areas that are heterogeneous with respect to morphology and molecular features. We established a method for cryopreservation of entire prostates from cancer patients, and build tissue microarrays from individual cancerous prostates representing >30 different areas of the primary tumor as well as multiple lymph node metastases. We analyzed these arrays for ERG fusion by FISH and for peptide signatures by MALDI MSI. In addition, 2 selected tumor areas and 4 different lymph node metastases from one prostate were subjected to paired end next generation sequencing. MALDI identified 5 masses that were specific for epithelial cells. One particular mass was strongly related to presence of ERG fusion. In one prostate, we identified another mass that was present only in 2/35 tissue spots from the primary tumor as well as in 4/10 lymph nodes. Deep sequencing of different tumor areas of this case identified distinct patterns of structural genetic alterations. A cluster analysis showed that all samples were clonally related, and identified the area inside the primary tumor that gave rise to the metastases. In conclusion, these data suggest that MALDI imaging and next generation sequencing allow for identification of genetic and proteome patterns that correspond to metastatic spread of prostate cancer cells. A combination of both techniques is suited to dissect the genetic background of distinct MALDI signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1272. doi:1538-7445.AM2012-1272

PurposeTo develop a general tissue preparation protocol for MALDI (Matrix-Assisted Laser Desorption Ionization) imaging mass spectrometry of ocular lens tissue, and to compare the spatial distributions of α-crystallin and its modified forms in bovine and rabbit lenses.MethodsFrozen bovine and rabbit lenses were cryosectioned equatorially at −20 °C into 12 μm-thick tissue sections. Lens sections were mounted onto conductive glass slides by ethanol soft-landing to maintain tissue integrity. An ethanol/xylene washing procedure was applied to each section before matrix application to facilitate uniform matrix crystal formation across the entire tissue section. Molecular images of both α-crystallin subunits and their modified forms were obtained from mass spectral data acquired at 100 μm steps across both whole rabbit and half bovine lens sections.ResultsDistinct spatial patterns for the two subunits of α-crystallin and their modified forms were observed in the rabbit and bovine lens sections. While αA-crystallin was extensively degraded in the lens core of both species, rabbit lenses exhibited a greater degree of larger molecular weight truncation products. In contrast, αB-crystallin degradation was limited in both species. Interestingly, phosphorylation of αA- and αB-crystallin was most abundant in the middle cortex of both species.ConclusionsAn improved method for investigating the spatial distribution of α-crystallin in the ocular lens by MALDI imaging mass spectrometry has been developed. The localization of multiple degradation products and specific regions of α-crystallin phosphorylation in bovine and rabbit lenses gives new insight into the program of lens fiber cell differentiation and normal lens function.

The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with single cell spatial omics methods allows for a comprehensive investigation of single cell spatial information and matrisomal N-glycan and extracellular matrix protein imaging. Here, the performance of the antibody-directed single cell workflows coupled with MALDI-MSI are evaluated. Miralys™ photocleavable mass-tagged antibody probes (MALDI-IHC, AmberGen, Inc.), GeoMx DSP® (NanoString, Inc.), and Imaging Mass Cytometry (IMC, Standard BioTools Inc.) were used in series with MALDI-MSI of N-glycans and extracellular matrix peptides on formalin-fixed paraffin-embedded tissues. Single cell omics protocols were performed before and after MALDI-MSI. The data suggests that for each modality combination, there is an optimal order for performing both techniques on the same tissue section. An overall conclusion is that MALDI-MSI studies may be completed on the same tissue section as used for antibody-directed single cell modalities. This work increases access to combined cellular and extracellular information within the tissue microenvironment to enhance research on the pathological origins of disease.Graphical

The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).

Formalin-fixed neuroendocrine tissues from American cockroaches (Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histological localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample preparation protocol rendering the endogenous peptides readily amenable to MSI analysis. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aqueous washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly soluble are therefore retained in-tissue with this protocol. The method is fully “top-down”, that is, without laborious in situ enzymatic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepared P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally, in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 year old, we demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. Our findings put forward a useful method for digestion-free, high-throughput analysis of endogenous peptides from FFPE samples and offer the potential for reinvestigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) is transforming spatial molecular studies. However, applying MALDI-MSI to small, anatomically complex tissues remains challenging. One such structure is the cochlea, the auditory part of the inner ear that is critical for hearing. To address these challenges, we developed and implemented a streamlined workflow for sample preparation and processing to obtain MALDI-MSI data on mouse cochlea. Sample acquisition was optimized to minimize time and processing steps, allowing use of flash-frozen neonatal mouse heads. This workflow enabled high spatial resolution metabolomic and lipidomic imaging of the sagittally cryosectioned mouse cochlea using N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) matrix via sublimation. Optimized NEDC sublimation allowed high signal-to-noise, reduced delocalization, and salt tolerance, allowing acquisition of 5 μm-resolution imaging data on a MALDI-MSI instrument. Sublimation was found to be superior to spraying as a method for matrix application due to its higher signal-to-noise, particularly for lipids and fatty acids, and improved spatial resolution. Diverse metabolites and lipids were measured throughout the cochlear region, revealing distinct spatial distributions. Clustering identified reproducible physiological regions, including the otic capsule and spiral ducts. High spatial resolution imaging revealed distinct tissues, cell types, and molecular signatures within the cochlea. These findings establish the utility of high spatial resolution MALDI-MSI for auditory research, enabling molecular mapping of cochlear function and dysfunction.

Dietary habit is closely associated with healthspan. Functional food factors are key to maintaining a health metabolism in our bodies. Because functional food factors are main components to determine the quality of foods, many technologies have been established to analyze functional factors in foods. High-performance liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry is a solid approach to detect functional food factors with high sensitivity and specificity. Findings obtained from these mass spectrometric approaches play essential roles in estimating the quality of foods. However, these technologies are not available for the analysis of the spatial distribution of molecules of interest in foods. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is considered an ideal approach to visualize distribution of molecules in foods. MALDI-MSI is a two-dimensional MALDI-MS technology that can detect compounds in a tissue section without purification, separation, or labeling. MALDI-MSI can be used to visualize the spatial distribution of wide range of food components including protein, peptides, amino acids, lipids, carbohydrate, and vitamins. Although the methodology of MALDI-MSI in food science is not yet fully established, the versatility of MALDI-MSI is expected to open a new frontier in food science. In this mini review, we briefly summarized the applications of MALDI-MSI in the field of food science.

Bupleurum is a kind of medicinal plant that has made a great contribution to human health because of the presence of bioactive metabolites: Bupleurum saikosaponins and flavonoids. Despite their importance, it has been a challenge to visually characterize the spatial distribution of these metabolites in situ within the plant tissue, which is essential for assessing the quality of Bupleurum. The development of a new technology to identify and evaluate the quality of medicinal plants is therefore necessary. Here, the spatial distribution and quality characteristics of metabolites of three Bupleurum species: Bupleurum smithii (BS), Bupleurum marginatum var. stenophyllum (BM), and Bupleurum chinense (BC) were characterized by Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Twenty-nine metabolites, including saikosaponins, non-saikosaponins, and compounds from the saikosaponin synthesis pathway, were characterized. Some of these were successfully localized and visualized in the transverse section of roots. In these Bupleurum species, twelve saikosaponins, five non-saikosaponins, and five saikosaponin synthesis pathway compounds were detected. Twenty-two major influencing components, which exhibit higher ion intensities in higher quality samples, were identified as potential quality markers of Bupleurum. The final outcome indicates that BC has superior quality compared to BS and BM. MALDI-MSI has effectively distinguished the quality of these Bupleurum species, providing an intuitive and effective marker for the quality control of medicinal plants.

The incidence of osteoarthritis (OA) has been expected to increase due to an aging population, as well as an increased incidence of intra-articular (osteo-) chondral damage. Lipids have already been shown to be involved in the inflammatory process of OA. This study aims at revealing region-specific lipid profiles of the infrapatellar fat pad (IPFP) of OA or cartilage defect patients by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which could be used as biomarkers for early OA detection. A higher presence of phospholipids was found in OA patients compared with cartilage defect patients. In addition, a higher abundance of ether-linked phosphatidylethanolamines (PE O-s) containing arachidonic acid was specifically found in OA patients compared with cartilage defect patients. These lipids were mainly found in the connective tissue of the IPFP. Specific lipid species were associated to OA patients compared with cartilage defect patients. PE O-s have been suggested as possible biomarkers for OA. As these were found more abundantly in the connective tissue, the IPFP’s intra-tissue heterogeneity might play an important role in biomarker discovery, implying that the amount of fibrous tissue is associated with OA.Graphical

The spatial distribution of components in Moringa oleifera (Lam) seed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)

Significant advancement of mass spectrometry imaging for food chemistry

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful approach that has been widely used for in situ detection of various endogenous compounds in tissues. However, there are still challenges with in situ analysis of proteins using MALDI-MSI due to the ion suppression effects of small molecules in tissue sections. Therefore, tissue-washing steps are crucial for protein MALDI tissue imaging to remove these interfering molecules. Here, we successfully developed a new method named the concentration-descending washing strategy (CDWS) with methanol (MeOH), i.e., washing of biological tissue with 100%, 95%, and 70% MeOH solutions, for the enhancement of endogenous in situ protein detection and imaging in tissues using MALDI-MS. The method of MeOH-based CDWS (MeOH-CDWS) led to the successful in situ detection of 272 ± 3, 185 ± 4, and 134 ± 2 protein ion signals from rat liver, rat brain, and germinating Chinese-yew seed tissue sections, respectively. By comparison, 161 ± 2, 121 ± 1, and 114 ± 2 protein ions were detected by three commonly used methods, i.e., Carnoy's wash, ethanol (EtOH)-based CAWS (i.e., concentration-ascending washing strategy, 70% EtOH followed by 90% EtOH/9% AcOH), and isopropanol (iPrOH)-based CAWS (70% iPrOH followed by 95% iPrOH), respectively, in rat liver tissue sections, indicating that 68.9 ± 3.1%, 124.8 ± 3.3%, and 138.6 ± 4.4% more protein ion signals could be detected by the use of MeOH-CDWS than the three abovementioned washing strategies. Our results show that the use of MeOH-CDWS improves the performance of MALDI-MSI for in situ protein detection such as the number and intensity of proteins. The use of MeOH-CDWS improves the fixation of proteins and thus reduces the loss of proteins, which significantly reduces protein delocalization in tissue and enhances the performance of MALDI tissue imaging of protein. Thus, the use of MeOH-CDWS improves the quality of protein images in tissue sections through MALDI-MSI and has the potential to be used as standard practice for MALDI tissue imaging of proteins.

AB0769 Lipid Characterization of Chondrogenic Differentiation Using Maldi Mass Spectrometry Imaging

Visualizing the spatial distribution of functional metabolites in the root of Codonopsis pilosula: An investigation integrating metabolomics, serum pharmacochemistry, and MALDI-MSI.