Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly).

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 104-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.

Assessing Molecular Localization of Symbiont Microalgae in Coral Branches Through Mass Spectrometry Imaging.

Reef-building corals are a fundamental pillar of coral reef ecosystems in tropical and subtropical shallow environments. Corals harbor symbiotic dinoflagellates belonging to the family Symbiodiniaceae, commonly known as zooxanthellae. Extensive research has been conducted on this symbiotic relationship, yet the fundamental information about the distribution and localization of Symbiodiniaceae cells in corals is still limited. This information is crucial to understanding the mechanism underlying the metabolite exchange between corals and their algal symbionts, as well as the metabolic flow within holobionts. To examine the distribution of Symbiodiniaceae cells within corals, in this study, we used fluorescence imaging and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MS-Imaging) on branches of the Acropora tenuis coral. We successfully prepared frozen sections of the coral for molecular imaging without fixing or decalcifying the coral branches. By combining the results of MS-Imaging with that of the fluorescence imaging, we determined that the algal Symbiodiniaceae symbionts were not only localized in the tentacle and surface region of the coral branches but also inhabited the in inner parts. Therefore, the molecular imaging technique used in this study could be valuable to further investigate the molecular dynamics between corals and their symbionts.

The Mechanism of Ochratoxin Contamination of Artificially Inoculated Licorice Roots.

Ochratoxin (OT) contamination of medicinal herbs is a serious threat to human health. This study was performed to investigate the mechanism of OT contamination of licorice (Glycyrrhiza sp.) root. Licorice root samples were cut into eight parts, which were placed separately on sucrose-free Czapek Dox agar medium, inoculated with the spores of ochratoxigenic Aspergillus westerdijkiae. After incubation for 10 and 20 days, the OT contents of the samples were determined by high-performance liquid chromatography, and microtome sections prepared from the samples were analyzed by desorption electrospray ionization tandem mass spectrometry, to visualize OT localization. The same sections were further examined by light microscopy and scanning electron microscopy, to investigate the path of fungal mycelial penetration of the inner roots. OT concentrations tended to increase from the upper- to the middle-root parts. OTs were located in cut areas and areas of cork layer damage; they were not present in the undamaged cork layer, indicating that the structure of this layer prevents OT contamination of the licorice root.

Low-dose ethanol increases aflatoxin production due to the adh1-dependent incorporation of ethanol into aflatoxin biosynthesis.

Aflatoxins are toxic secondary metabolites produced by some aspergilli, including Aspergillus flavus. Recently, ethanol has attracted attention as an agent for the control of aflatoxin contamination. However, as aflatoxin biosynthesis utilizes acetyl coenzyme A, ethanol may be conversely exploited for aflatoxin production. Here, we demonstrated that not only the 13C of labeled ethanol, but also that of labeled 2-propanol, was incorporated into aflatoxin B1 and B2, and that ethanol and 2-propanol upregulated aflatoxin production at low concentrations (<1% and <0.6%, respectively). In the alcohol dehydrogenase gene adh1 deletion mutant, the 13C incorporation of labeled ethanol, but not labeled 2-propanol, into aflatoxin B1 and B2 was attenuated, indicating that the alcohols have different utilization pathways. Our results show that A. flavus utilizes ethanol and 2-propanol as carbon sources for aflatoxin biosynthesis and that adh1 indirectly controls aflatoxin production by balancing ethanol production and catabolism.

Desorption electrospray ionization-mass spectrometry imaging of carnitine and imidazole dipeptides in pork chop tissues.

Carnitine is essential for energy production and lipid metabolism in skeletal muscle. Carnosine and its methylated analogs anserine and balenine are histidine-containing imidazole dipeptides, which are antioxidative compounds. They are major health-related components in meat; however, analytical technique to investigate their distribution among tissues have not fully established. Here, we performed desorption electrospray ionization (DESI)-mass spectrometry imaging (MSI) of pork chop sections containing longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle to investigate the distributions of carnitine and imidazole dipeptides. Liquid chromatography-MS revealed that the concentrations of carnitine, carnosine, anserine, and balenine were 11.0 ± 0.9, 330.1 ± 15.5, 21.2 ± 1.5, and 9.6 ± 0.5 mg/100 g, respectively. In the mass spectrum obtained by DESI-MSI, peaks corresponding to the chemical formulae of carnitine and imidazole dipeptides were detected. DESI-MSI provided definite identification of carnitine, while DESI-tandem MSI (MS/MSI) was necessary to accurately visualize carnosine, anserine, and balenine. Carnitine and these imidazole dipeptides were mainly distributed in the loin and spinalis muscle, while their distribution was not uniform in both muscle tissues. In addition, the balance between both tissues were different. The concentration of carnitine was higher in the spinalis muscle than that in the loin, while those of imidazole dipeptides were higher in the loin than those in the spinalis muscle. These results were consistent with those obtained by liquid chromatography-MS quantification, suggest that DESI-MSI analysis is useful for the distribution analysis of carnitine and imidazole dipeptides in meat.

Eicosapentaenoic acid is associated with the attenuation of dysfunctions of mesenchymal stem cells in the abdominal aortic aneurysm wall.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by progressive dilation of the aorta which is reportedly associated with inflammation. Previous studies suggested that eicosapentaenoic acid (EPA) has suppressive effects on AAA development via anti-inflammatory activities. However, relationships between the anti-inflammatory effects and the cells in the AAA wall are poorly understood. In this study, we visualized the distribution of EPA-containing phosphatidylcholine (EPA-PC) in the AAA wall. EPA-PC was not ubiquitously distributed in both animal (hypoperfusion-induced AAA model) and human AAA walls, suggesting the preferential incorporation of EPA into certain cells. In the EPA-PC-high region of both animal and human AAAs, mesenchymal stem cell (MSC) marker positive areas were significantly higher than those in the EPA-PC-low region. Matrix metalloproteinase-positive MSCs were significantly lower in the AAA wall of the animal model which was administered EPA-rich fish oil. These data suggest that EPA is associated with the attenuation of MSC dysfunctions, which result in the suppression of AAA development.

A new metabolite, mannogeranylnerol, specifically produced at body temperature by Schizophyllum commune, a causative fungus of human mycosis.

Schizophyllum commune is a causative fungus of human mycosis. Its metabolites produced at 27 °C were compared with those produced at 37 °C, to obtain a candidate low-molecular-weight virulence factor related to the pathogenicity of this fungus. We found that S. commune specifically produces two acyclic terpene mannosides at 37 °C. They were identified as nerolidol ß-D-mannoside (1) and geranylnerol ß-D-mannoside (2) by NMR, MS, and CD analyses. Compound 2, a new compound named mannogeranylnerol, showed weak antibiotic activity that was slightly stronger than that of compound 1.

Distribution analysis of jasmonic acid-related compounds in developing Glycine max L. (soybean) seeds using mass spectrometry imaging and liquid chromatography-mass spectrometry.

INTRODUCTION: Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA) and hexadecatrienoic acid, and regulate seed development. However, their spatial distribution in the developing Glycine max L. (soybean) seeds has not been elucidated. OBJECTIVE: To investigate the distribution of JA-related compounds in the developing soybean seeds using desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) analyses. METHODS: Cryosections of developing seeds were prepared using adhesive films, and subjected to DESI-MSI analysis. Verification of the DESI-MSI ion images were performed using DESI-tandem MSI (MS/MSI), LC-ESI-MS and tandem MS (MS/MS). RESULTS: In the DESI-MSI mass spectrum, peaks matching the chemical formulae of αLA, 12-oxo-phytodienoic acid (OPDA), and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were detected. These compounds were mainly distributed in the seed coat, especially near the hilum. This was consistent with the quantitative results obtained by LC-ESI-MS. While, DESI-MS/MSI and LC-ESI-MS/MS suggested the presence of isomers for OPDA and OPC-8:0. The effect of isomers on the DESI-MSI ion images was small for OPDA, and considerable for OPC-8:0. CONCLUSION: These results demonstrated that free αLA, OPDA, and OPC-8:0 were the abundant JA-related compounds mainly distributed in the seed coat of the developing soybeans. OPDA and OPC-8:0 might exert a biological role in the seed coat. To the best of my knowledge, this is the first report on the accumulation of OPDA and OPC-8:0 in the seed coat. The combination of DESI-MSI and LC-ESI-MS is a useful tool for distribution analysis of JA-related compounds in the developing seeds.

Unique distribution of ellagitannins in ripe strawberry fruit revealed by mass spectrometry imaging.

Ellagitannins (ETs) are hydrolysable tannins composed of a polyol core, primarily glucose, which is esterified with hexahydroxydiphenic acid (HHDP), and in some cases, gallic acid. ETs are the major phenolic compounds found in strawberries and may contribute to the health-related properties of strawberries, because of their strong antioxidative activity. However, their distribution in the strawberry fruit remains unclear. In this study, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize ETs in ripe strawberry fruits. Five peaks, corresponding to the m/z values of ET [M-H]- ions detected in the MALDI-MS spectrum of strawberry extracts, were identified as strictinin, pedunculagin, casuarictin, davuriicin M1, and an unknown ET using MALDI-tandem MS (MS/MS). In addition, liquid chromatography-electrospray ionization-MS/MS of the extracts revealed the presence of pedunculagin isomers and the unknown ET. Ion images of these five ETs were reconstructed using MALDI-MSI. Strictinin was widely distributed in and around the achene seed coats, while the other ETs were dispersed in and around the seed coats, and at the bottom of the receptacle; pedunculagin was distributed in the epidermis and pith, whereas casuarictin, the unknown ET, and davuriicin M1 were distributed in the pith. Moreover, MALDI-MSI of a casuarictin standard indicated that in-source fragmentation weakly affected the ion images. The results suggest that the distribution of ETs depends on the presence or absence of their constituents, namely galloyl units, HHDP, and bis-HHDP. To the best of my knowledge, this is the first report on the visualization of ETs in plant tissues using MSI, MALDI-MSI may be a useful tool for analyzing the distribution of ETs in the strawberry fruit.

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.

Unique localization of jasmonic acid-related compounds in developing Phaseolus vulgaris L. (common bean) seeds revealed through desorption electrospray ionization-mass spectrometry imaging.

Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA). Presumably, they are involved in the regulation of seed embryogenesis, dormancy, and germination. However, their spatial localization in the developing Phaseolus vulgaris L. (common bean) seeds has not been fully elucidated. Therefore, desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was performed to investigate their localization in the developing seeds. Peaks corresponding to the chemical formulae of αLA and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were localized mainly in the radicle and seed coat, while that of 12-oxo-phytodienoic acid (OPDA) in the seed coat. This was consistent with the quantitative results obtained using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis. In contrast, DESI-tandem MSI (MS/MSI) and LC-ESI-MS/MS analyses showed that the effects of isomers on the DESI-MSI ion images were small for αLA and OPDA, but not for OPC-8:0. This indicated that DESI-MSI could accurately visualize αLA and OPDA, while DESI-MS/MSI was necessary to visualize OPC-8:0. The results demonstrated that free αLA and OPC-8:0 were abundant in the radicle and seed coat, while free OPDA was accumulated in the seed coat. Interestingly, the localization pattern of OPDA was similar to that of JA. In addition, compared to the concentrations of OPDA, the concentration of OPC-8:0 was lower in the seed coat and higher in the radicle. These results suggest that OPDA and/or JA play a biological role mainly in the seed coat, while OPC-8:0 is biologically active mainly in the radicle. Therefore, DESI-MSI coupled with LC-ESI-MS is a useful tool for spatial analysis of JA-related compounds in developing common bean seeds.

Similar distribution of orally administered eicosapentaenoic acid and M2 macrophage marker in the hypoperfusion-induced abdominal aortic aneurysm wall.

Abdominal aortic aneurysm (AAA) is an aortic disease in which the aortic diameter is ≥3.0 cm; if left untreated, the aortic wall continues to weaken, resulting in progressive dilatation. Effective therapeutic drugs for AAA patients have not been discovered. Eicosapentaenoic acid (EPA) reportedly attenuates the development of AAA in experimental AAA animal models. However, the underlying mechanism of action is still not totally clear. To understand the mechanism, we visualized the distribution of EPA-containing phosphatidylcholine (PC) in the AAA wall by matrix-assisted laser desorption ionization-mass spectrometry imaging. EPA-containing PC was characteristically distributed in the AAA wall, and the positive area for the M2 macrophage marker was significantly higher in the region where EPA-containing PC was highly detected (region 2) than in the region where EPA-containing PC was poorly detected (region 1). The M1 macrophage marker levels were not different between regions 1 and 2. A comparative observation showed a similar distribution of the M2 macrophage marker and EPA-containing PC. These data suggest the preferential incorporation of EPA into M2 macrophages. Positive areas for matrix metalloproteinase 2 and malondialdehyde in region 2 were significantly lower than those in region 1. The reported suppressive effect of EPA on the development of AAA may be partly attributed to the increased anti-inflammatory property of M2 macrophages.

Adhesive film applications help to prepare strawberry fruit sections for desorption electrospray ionization-mass spectrometry imaging.

Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) is a powerful tool to analyze the distribution of metabolites in biological tissues. Cryosectioning of biological tissues is usually required prior to DESI-MSI, but it can be difficult for tissues that are fragile, hard, and have a high-water content. The Kawamoto method uses transparent adhesive films to prepare cryosections; however, its application for plant tissues, such as strawberry tissues, in DESI-MSI has not been verified. In this study, strawberry cryosections maintained original structures were prepared using adhesive film. Subsequently, numerous peaks were detected for the sections using the positive and negative ion modes of DESI-MSI. Several primary and specialized metabolites, such as amino acids, sugars, organic acids, and flavonoids, were identified and visualized. These results suggest the use of adhesive films when cryosectioning could improve DESI-MSI analysis of the metabolites in strawberry fruits and various tissues of other plant species.

Direct LC-ESI-MS/MS analysis of plant glucosylceramide and ceramide species with 8E and 8Z isomers of the long chain base.

Glucosylceramides and ceramides with 8E and 8Z isomers of the long chain base are found in plants. These isomers have been difficult to quantify separately using liquid chromatography-tandem mass spectrometry (LC-MS/MS) because the isomers have the same retention time, their precursor and product ions have the same m/z values, and plant ceramide standards are not commercially available. Here we tested trial separations using various ODS columns and prepared plant ceramide standards generated by human glucocerebrosidase (imiglucerase) using commercially available plant glucosylceramide standards as the substrates. Consequently, we were able to quantify the isomers based on differences in retention times in a TSKgel ODS-120A column (Tosoh, Tokyo Japan) using LC-electrospray ionization-MS/MS (LC-ESI-MS/MS).

Mass Spectrometry Imaging of Flavonols and Ellagic Acid Glycosides in Ripe Strawberry Fruit.

Flavonols and ellagic acid glycosides are major phenolic compounds in strawberry fruit. They have antioxidant activity, show protective functions against abiotic and biotic stress, and provide health benefits. However, their spatial distribution in ripe fruit has not been understood. Therefore, matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) was performed to investigate their distribution in fruit tissues. Using strawberry extract, five flavonols, namely, three kaempferols and two quercetins, and two ellagic acid glycosides, were tentatively identified by MALDI-tandem MS. To investigate the tentatively identified compounds, MALDI-MSI and tandem MS imaging (MS/MSI) analyses were performed. Kaempferol and quercetin glycosides showed similar distribution patterns. They were mainly found in the epidermis, while ellagic acid glycosides were mainly found in the achene and in the bottom area of the receptacle. These results suggested that the difference in distribution pattern between flavonols and ellagic acid glycosides depends on the difference between their aglycones. Seemingly, flavonols play a role in protective functions in the epidermis, while ellagic acid glycosides play a role in the achene and in the bottom side of the receptacle, respectively. These results demonstrated that MALDI-MSI is useful for distribution analysis of flavonols and ellagic acid glycosides in strawberry fruit.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Isolation, Evaluation, and Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides from Game Meat.

Game meat has been underutilized, while it offers the potential to diversify not only the human diet but also increase food production and the nutritional value of meat products. This study aimed to determine the angiotensin I-converting enzyme (ACE) inhibitory activities of the digested game meats (venison and boar meat) compared with those of livestock meats (beef and pork). Through the sodium dodecyl sulfate polyacrylamide gel electrophoresis and size chromatography results, we found that the digested products from each meat had different molecular weights. The ACE inhibitory ratio in all tested samples had gradually increased following by the enzyme treatments. ACE inhibitory ratios and the half maximal inhibitory concentration values indicated that digested venison was the most potent inhibitor of ACE activity, followed by the digested boar meat. The level of anserine in digested venison was higher than that in the other meats, but the carnosine level was lower. Through fractionations and liquid chromatography-tandem mass spectrometry analysis, five ACE inhibitory peptides were identified from the digested venison. Of these peptides, Isoleucine-Lysine- Glutamic Acid-Valine-Threonine-Glutamic Acid-Arginine (IKEVTER) demonstrated the highest ACE inhibitory activity. Therefore, the game meat is food that is believed potentially to offer high bioactivities, particularly antihypertensive forces.

Effects of whey protein hydrolysate on growth promotion and immunomodulation in mouse pups in artificial rearing system.

This study aimed to investigate the effects of whey protein hydrolysate (WPH) on the growth and immunity of mouse pups in artificial rearing (AR) system. Mouse pups were reared in the AR system with artificial milk including 5% WPH (AR with WPH) or not (AR without WPH), and the remaining pups were reared by their mother (dam) for 14 days after birth. The body weight change and body weight gain rates in the AR with WPH group were significantly higher than those observed in the AR without WPH group and similar to those in the dam group. Moreover the feed and protein efficiencies in the AR with WPH group were significantly higher than those of the AR without WPH group. In addition, the supplement of WPH in the AR system was shown to significantly elevate the number of CD3+ CD8+ , B220+ CD19+ , IA/IE+ CD11c+ , and CD11b+ in the thymocyte and/or splenocyte, and the thymus weight. Furthermore, MALDI-TOF/MS analysis identified the amino acid sequences corresponding to some peptides, and indicated that VRTPEVDDE had the highest relative intensity among the peptides from tested WPH. Therefore, WPH would be required to not only promote growth, but also exert immunomodulatory activities in mouse pups in AR system.

Localization of Flavan-3-ol Species in Peanut Testa by Mass Spectrometry Imaging.

Flavan-3-ols, procyanidins and their monomers are major flavonoids present in peanuts that show a wide range of biological properties and health benefits, based on their potent antioxidant activity. Procyanidin oligomers, especially A-type, are reportedly abundant in peanut skin; however, their localization in the raw peanut testa remains poorly understood. Therefore, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to investigate the localization of flavan-3-ols in peanut testa. 1,5-Diaminonaphthalene was coated onto the peanut section by matrix vapor deposition/recrystallization, and MALDI-MSI measurements were performed in the negative-ion mode. Peaks matching the m/z values of flavan-3-ol [M - H]- ions were observed in the mass spectrum extracted from the outer epidermis of the peanut testa, using the region of interest function. Catechin and/or epicatechin, five A-type, and one B-type procyanidins were assigned by the fragment ions generated by retro-Diels-Alder, heterocyclic ring fission, and quinone methide reactions detected in MALDI-tandem MS spectra. These flavan-3-ols were localized in the outer epidermis of the peanut testa. This information will contribute to improving the extraction and purification efficiencies of flavan-3-ols from peanut testa. As flavan-3-ols display anti-microbial activity, it is speculated that flavan-3-ols present in the outer epidermis of peanut testa act to prevent pathogen infection.

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.