ABSTRACT
Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response.
Subject(s)
Arabidopsis , Phytophthora infestans , Solanum tuberosum , Solanum tuberosum/metabolism , Peptides/pharmacology , Peptides/metabolism , Arabidopsis/metabolism , Phytophthora infestans/physiology , Immunity , Amino Acids/metabolism , Plant Diseases/geneticsABSTRACT
The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions.
Subject(s)
Arabidopsis Proteins , Arabidopsis , ATP-Binding Cassette Transporters/metabolism , Arabidopsis/metabolism , Thiazoles/metabolism , Phytoalexins , Arabidopsis Proteins/metabolism , Plant Diseases/microbiology , Gene Expression Regulation, PlantABSTRACT
Glyphosate, the most successful herbicide in history, specifically inhibits the activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), one of the key enzymes in the shikimate pathway. Amaranthus palmeri is a driver weed in agriculture today that has evolved glyphosate-resistance through increased EPSPS gene copy number and other mechanisms. Non-targeted GC-MS and LC-MS metabolomic profiling was conducted to examine the innate physiology and the glyphosate-induced perturbations in one sensitive and one resistant (by EPSPS amplification) population of A. palmeri. In the absence of glyphosate treatment, the metabolic profile of both populations was very similar. The comparison between the effects of sublethal and lethal doses on sensitive and resistant populations suggests that lethality of the herbicide is associated with an amino acid pool imbalance and accumulation of the metabolites of the shikimate pathway upstream from EPSPS. Ferulic acid and its derivatives were accumulated in treated plants of both populations, while quercetin and its derivative contents were only lower in the resistant plants treated with glyphosate.
ABSTRACT
Late blight, caused by the oomycete Phytophthora infestans, is economically the most important foliar disease of potato. To assess the importance of the leaf surface, as the site of the first encounter of pathogen and host, we performed untargeted profiling by liquid chromatography-mass spectrometry of leaf surface metabolites of the susceptible cultivated potato Solanum tuberosum and the resistant wild potato species Solanum bulbocastanum. Hydroxycinnamic acid amides, typical phytoalexins of potato, were abundant on the surface of S. tuberosum, but not on S. bulbocastanum. One of the metabolites accumulating on the surface of the wild potato was identified as lysophosphatidylcholine carrying heptadecenoic acid, LPC17:1. In vitro assays revealed that both spore germination and mycelial growth of P. infestans were efficiently inhibited by LPC17:1, suggesting that leaf surface metabolites from wild potato species could contribute to early defense responses against P. infestans.
Subject(s)
Phytophthora infestans , Solanum tuberosum , Solanum , Lysophosphatidylcholines , Plant Diseases , Plant LeavesABSTRACT
Plants exude a diverse cocktail of metabolites into the soil as response to exogenous and endogenous factors. So far, root exudates have mainly been studied under artificial conditions due to methodological difficulties. In this study, each five perennial grass and forb species were investigated for polar and semi-polar metabolites in exudates under field conditions. Metabolite collection and untargeted profiling approaches combined with a novel classification method allowed the designation of 182 metabolites. The composition of exuded polar metabolites depended mainly on the local environment, especially soil conditions, whereas the pattern of semi-polar metabolites was primarily affected by the species identity. The profiles of both polar and semi-polar metabolites differed between growth forms, with grass species being generally more similar to each other and more responsive to the abiotic environment than forb species. This study demonstrated the feasibility of investigating exudates under field conditions and to identify the driving factors of exudate composition.
Subject(s)
Plant Exudates/metabolism , Plant Roots/metabolism , Poaceae/metabolism , Asteraceae/classification , Asteraceae/metabolism , Ecosystem , Gas Chromatography-Mass Spectrometry , Grassland , Plant Physiological Phenomena , Plantaginaceae/classification , Plantaginaceae/metabolism , Poaceae/classification , Ranunculaceae/classification , Ranunculaceae/metabolism , Rhizosphere , Rubiaceae/classification , Rubiaceae/metabolismABSTRACT
Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self-maintaining phytohormone salicylic acid (SA)-defense loop. In spatial and temporal resolution, we show that calcium-dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance, CPK5 functions upstream of SA synthesis, perception, and signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR-inducing metabolite N-hydroxy-L-pipecolic acid (NHP) and SAR marker genes, including Systemic Acquired Resistance Deficient 1 (SARD1) Plants of increased CPK5, but not CPK6, signaling display an 'enhanced SAR' phenotype towards a secondary bacterial infection. In the sard1-1 background, CPK5-mediated basal resistance is still mounted, but NHP concentration is reduced and enhanced SAR is lost. The biochemical analysis estimated CPK5 half maximal kinase activity for calcium, K50 [Ca2+ ], to be c. 100 nM, close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium, a prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity.
Subject(s)
Arabidopsis Proteins/metabolism , Calcium Signaling , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Disease Resistance/immunology , Immunologic Memory , Pipecolic Acids/metabolism , Plant Diseases/immunology , Plant Immunity , Salicylic Acid/metabolism , Calcium/metabolism , Disease Resistance/genetics , Gene Expression Regulation, Plant , Genes, Plant , Immunologic Memory/genetics , Plant Diseases/genetics , Plant Immunity/geneticsABSTRACT
Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.
Subject(s)
Gene Expression Regulation, Plant/immunology , Phytophthora infestans/pathogenicity , Plant Diseases/immunology , Plant Proteins/immunology , Protein Serine-Threonine Kinases/immunology , Receptors, Pattern Recognition/immunology , Solanum tuberosum/immunology , Alkaloids/immunology , Alkaloids/metabolism , Amides/immunology , Amides/metabolism , Coumaric Acids/immunology , Coumaric Acids/metabolism , Cyclopentanes/immunology , Cyclopentanes/metabolism , Disease Resistance/genetics , Flavonoids/immunology , Flavonoids/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/immunology , Metabolome/genetics , Metabolome/immunology , Oxylipins/immunology , Oxylipins/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology , Phytophthora infestans/physiology , Plant Diseases/genetics , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Plants, Genetically Modified , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism , Receptors, Pattern Recognition/antagonists & inhibitors , Receptors, Pattern Recognition/genetics , Salicylic Acid/immunology , Salicylic Acid/metabolism , Sesquiterpenes/immunology , Sesquiterpenes/metabolism , Solanum tuberosum/genetics , Solanum tuberosum/parasitology , PhytoalexinsABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0204128.].
ABSTRACT
Bryophytes occur in almost all land ecosystems and contribute to global biogeochemical cycles, ecosystem functioning, and influence vegetation dynamics. As growth and biochemistry of bryophytes are strongly dependent on the season, we analyzed metabolic variation across seasons with regard to ecological characteristics and phylogeny. Using bioinformatics methods, we present an integrative and reproducible approach to connect ecology with biochemistry. Nine different bryophyte species were collected in three composite samples in four seasons. Untargeted liquid chromatography coupled with mass spectrometry (LC/MS) was performed to obtain metabolite profiles. Redundancy analysis, Pearson's correlation, Shannon diversity, and hierarchical clustering were used to determine relationships among species, seasons, ecological characteristics, and hierarchical clustering. Metabolite profiles of Marchantia polymorpha and Fissidens taxifolius which are species with ruderal life strategy (R-selected) showed low seasonal variability, while the profiles of the pleurocarpous mosses and Grimmia pulvinata which have characteristics of a competitive strategy (C-selected) were more variable. Polytrichum strictum and Plagiomnium undulatum had intermediary life strategies. Our study revealed strong species-specific differences in metabolite profiles between the seasons. Life strategies, growth forms, and indicator values for light and soil were among the most important ecological predictors. We demonstrate that untargeted Eco-Metabolomics provide useful biochemical insight that improves our understanding of fundamental ecological strategies.
ABSTRACT
Primary and secondary metabolites exuded by plant roots have mainly been studied under laboratory conditions, while knowledge of root exudate patterns of plants growing in natural communities is very limited. Focusing on ten common European grassland plant species, we asked to which degree exuded metabolite compositions are specific to species or growth forms (forbs and grasses), depend on environments and local neighbourhoods, and reflect traditional plant functional traits. Root exudates were collected under field conditions and analysed using a non-targeted gas chromatography coupled mass spectrometry (GC-MS) approach. In total, we annotated 153 compounds of which 36 were identified by structure and name as metabolites mainly derived from the primary metabolism. Here we show by using variance partitioning, that the composition of exuded polar metabolites was mostly explained by plot identity, followed by plant species identity while plant species composition of the local neighbourhood played no role. Total and root dry biomass explained the largest proportion of variance in exudate composition, with additional variance explained by traditional plant traits. Although the exudate composition was quite similar between the two growth forms, we found some metabolites that occurred only in one of the two growth forms. Our study demonstrated the feasibility of measuring polar exudates under non-sterile field conditions by mass spectrometry, which opens new avenues of research for functional plant ecology.
Subject(s)
Plant Exudates/chemistry , Plant Roots/metabolism , Plants/metabolism , Poaceae/metabolism , Gas Chromatography-Mass Spectrometry , Grassland , Seedlings/growth & development , Seeds/growth & development , Water/chemistryABSTRACT
In Eco-Metabolomics interactions are studied of non-model organisms in their natural environment and relations are made between biochemistry and ecological function. Current challenges when processing such metabolomics data involve complex experiment designs which are often carried out in large field campaigns involving multiple study factors, peak detection parameter settings, the high variation of metabolite profiles and the analysis of non-model species with scarcely characterised metabolomes. Here, we present a dataset generated from 108 samples of nine bryophyte species obtained in four seasons using an untargeted liquid chromatography coupled with mass spectrometry acquisition method (LC/MS). Using this dataset we address the current challenges when processing Eco-Metabolomics data. Here, we also present a reproducible and reusable computational workflow implemented in Galaxy focusing on standard formats, data import, technical validation, feature detection, diversity analysis and multivariate statistics. We expect that the representative dataset and the reusable processing pipeline will facilitate future studies in the research field of Eco-Metabolomics.
Subject(s)
Bryophyta/metabolism , Metabolomics/methods , Secondary Metabolism , Bryophyta/genetics , Metabolome , SeasonsABSTRACT
Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected â¼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.
Subject(s)
Formaldehyde/metabolism , Metabolomics/methods , Paraffin Embedding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tissue Fixation , Aminacrine/metabolism , Fourier Analysis , Humans , Molecular WeightABSTRACT
The identification of metabolites by mass spectrometry constitutes a major bottleneck which considerably limits the throughput of metabolomics studies in biomedical or plant research. Here, we present a novel approach to analyze metabolomics data from untargeted, data-independent LC-MS/MS measurements. By integrated analysis of MS(1) abundances and MS/MS spectra, the identification of regulated metabolite families is achieved. This approach offers a global view on metabolic regulation in comparative metabolomics. We implemented our approach in the web application "MetFamily", which is freely available at http://msbi.ipb-halle.de/MetFamily/ . MetFamily provides a dynamic link between the patterns based on MS(1)-signal intensity and the corresponding structural similarity at the MS/MS level. Structurally related metabolites are annotated as metabolite families based on a hierarchical cluster analysis of measured MS/MS spectra. Joint examination with principal component analysis of MS(1) patterns, where this annotation is preserved in the loadings, facilitates the interpretation of comparative metabolomics data at the level of metabolite families. As a proof of concept, we identified two trichome-specific metabolite families from wild-type tomato Solanum habrochaites LA1777 in a fully unsupervised manner and validated our findings based on earlier publications and with NMR.
Subject(s)
Metabolome , Metabolomics , Chromatography, High Pressure Liquid , Cluster Analysis , Solanum lycopersicum/metabolism , Magnetic Resonance Spectroscopy , Plant Leaves/metabolism , Principal Component Analysis , Tandem Mass Spectrometry , User-Computer InterfaceABSTRACT
MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals' germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest.
Subject(s)
Germination , Hordeum/metabolism , Plant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Antifungal Agents/metabolism , Cluster Analysis , Computational Biology , Endosperm/metabolism , Lipids/chemistry , Oligosaccharides/metabolism , Phosphatidylcholines/metabolismABSTRACT
The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface.
Subject(s)
Arabidopsis/metabolism , Coumaric Acids/metabolism , Disease Resistance , Gene Expression Regulation, Plant , Phytophthora infestans/physiology , Plant Diseases/immunology , Amides/metabolism , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/microbiology , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/metabolism , Plant Leaves/microbiology , Plants, Genetically Modified , Solanum tuberosum/microbiologyABSTRACT
We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.
Subject(s)
Biomarkers, Tumor/metabolism , Fixatives/chemistry , Formaldehyde/chemistry , Metabolomics/methods , Neoplasms/metabolism , Paraffin Embedding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tissue Fixation/methods , Cluster Analysis , Computational Biology , Cyclotrons , Diagnosis, Differential , Disease-Free Survival , Female , Fourier Analysis , Germany , Humans , Male , Neoplasms/mortality , Neoplasms/pathology , Neoplasms/therapy , Netherlands , Predictive Value of Tests , Proportional Hazards Models , Reproducibility of Results , Time Factors , Treatment OutcomeABSTRACT
Salt stress impairs global agricultural crop production by reducing vegetative growth and yield. Despite this importance, a number of gaps exist in our knowledge about very early metabolic responses that ensue minutes after plants experience salt stress. Surprisingly, this early phase remains almost as a black box. Therefore, systematic studies focussing on very early plant physiological responses to salt stress (in this case NaCl) may enhance our understanding on strategies to develop crop plants with a better performance under saline conditions. In the present study, hydroponically grown Vicia faba L. plants were exposed to 90 min of NaCl stress, whereby every 15 min samples were taken for analyzing short-term physiologic responses. Gas chromatography-mass spectrometry-based metabolite profiles were analysed by calculating a principal component analysis followed by multiple contrast tests. Follow-up experiments were run to analyze downstream effects of the metabolic changes on the physiological level. The novelty of this study is the demonstration of complex stress-induced metabolic changes at the very beginning of a moderate salt stress in V. faba, information that are very scant for this early stage. This study reports for the first that the proline analogue trans-4-hydroxy-L-proline, known to inhibit cell elongation, was increasingly synthesized after NaCl-stress initiation. Leaf metabolites associated with the generation or scavenging of reactive oxygen species (ROS) were affected in leaves that showed a synchronized increase in ROS formation. A reduced glutamine synthetase activity indicated that disturbances in the nitrogen assimilation occur earlier than it was previously thought under salt stress.
Subject(s)
Nitrogen/metabolism , Proline/biosynthesis , Reactive Oxygen Species/metabolism , Salt Tolerance , Sodium Chloride/metabolism , Stress, Physiological , Vicia faba/metabolism , Gas Chromatography-Mass Spectrometry , Glutamine/metabolism , Plant Leaves/metabolism , Plant Proteins/metabolism , Plant Roots/metabolism , Proline/analogs & derivativesABSTRACT
The molecular investigation of archived formalin-fixed, paraffin-embedded (FFPE) tissue samples provides the chance to obtain molecular patterns as indicatives for treatment and clinical end points. MALDI mass spectrometry imaging is capable of localizing molecules like proteins and peptides in tissue sections and became a favorite platform for the targeted and non-targeted approaches, especially in clinical investigations for biomarker research. In FFPE tissues the recovery of proteomic information is constrained by fixation-induced cross-links of proteins. The promising new insights obtained from FFPE in combination with the comprehensive patients' data caused much progress in the optimization of MS imaging protocols to investigate FFPE samples. This review presents the past and current research in MALDI MS imaging of FFPE tissues, demonstrating the improvement of analyses, their actual limitations, but also the promising future perspectives for histopathological and tissue-based research.
Subject(s)
Formaldehyde/chemistry , Paraffin Embedding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Biomarkers/chemistry , Cross-Linking Reagents/chemistry , Humans , Peptides/chemistry , Phylogeny , Proteins/chemistry , Proteomics , Tissue FixationABSTRACT
Mass spectrometry imaging was applied on germinated barley for the detailed localization of metabolites in longitudinal and transversal seed sections. Among others, 20 m/z signals occurred in three regular peak clusters with specific, distinct localizations in embryo tissues. High resolution FT-ICR MS, MALDI-TOF MS/MS, and UHPLC-ESI MS/MS served for the identification and structural characterization of these compounds. Only five metabolites were published in their structures, namely the antifungal compounds hordatine A and B in non-glycosylated and glycosylated forms. All other non-identified cluster compounds were of hordatine-like structure and differed by systematic O-methylations, hydroxylations, and glycosylations. These differences in molecular structures correlated to distinct localization patterns within the embryo and might serve for the regulation of antifungal properties. Based on the structural investigations by mass spectrometry, an array of different hordatines that comprises the five published hordatines, 15 novel hordatine derivates and their six precursors could be localized in the embryo of germinated barley. Implications for the biosynthetic pathway and transport processes are discussed.