Matrix-assisted Laser Desorption Ionization Mass Research Articles

Insight to Functional Conformation and Noncovalent Interactions of Protein-Protein Assembly Using MALDI Mass Spectrometry.

Noncovalent interactions are the keys to the structural organization of biomolecule e.g., proteins, glycans, lipids in the process of molecular recognition processes e.g., enzyme-substrate, antigen-antibody. Protein interactions lead to conformational changes, which dictate the functionality of that protein-protein complex. Besides biophysics techniques, noncovalent interaction and conformational dynamics, can be studied via mass spectrometry (MS), which represents a powerful tool, due to its low sample consumption, high sensitivity, and label-free sample. In this review, the focus will be placed on Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and its role in the analysis of protein-protein noncovalent assemblies exploring the relationship within noncovalent interaction, conformation, and biological function.

In Situ Proteomic Analysis of Glioblastoma Multiforme: A Translational Approach to Improve Prognostic/Diagnostic Routines

In order to evaluate the use of matrix-assisted laser desorption ionization (MALDI) Mass Spectral Imaging (MSI) to Glioblastoma (GBM) studies, some sections of formalin fixed paraffin embedded samples of GBM tumors were submitted to classical immunoassays, to define the profile of distribution of some classical and well recognized molecular markers of GBM grade IV (Ki-67, S100, Glial GFAP, CD31 and CD34), while other sections of the same samples were submitted to in-tissue proteomic analysis by MALDI MSI, and both results compared to each other. The overlapping of the MALDI spectra obtained for the tryptic peptides with the immunohistochemical reactions of each marker protein were used to build a distribution map of the marker proteins all over the GBM tissue section. The results revealed a high correlation between both methods, indicating that MALDI MSI has enough sensitivity to be compared to the immunohistochemical methods, as well is sufficiently reliable to be used in biomarkers identification.

MALDI-MSI-A Step Forward in Overcoming the Diagnostic Challenges in Ovarian Tumors.

This study presents the use of matrix-assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) directly on the tissue of two ovarian tumors that often present a diagnostic challenge, a low-grade serous borderline ovarian tumor and ovarian fibrothecoma. Different spatial distribution of m/z values within the tissue samples was observed, and regiospecific peaks were identified. Among the 106 peaks in the borderline ovarian tumor five, regiospecific peaks (m/z: 2861.35; 2775.79; 3368.34; 3438.43; 4936.37) were selected using FlexImaging software. Subsequently, the distribution of those selected peaks was visualized on the fibrothecoma tissue section, which demonstrated the differences in the tissue homo-/heterogeneous structure of both tumors. The comparison with the histopathological staining of the ovarian borderline tumor tissue section, obtained during serial sectioning, showed a close correlation of the molecular map with the morphological and histopathological features of the tissue and allowed the identification of different tissue types within the sample. This study highlights the potential significance of MSI in enabling morphological characterization of ovarian tumors as well as correct diagnosis and further prognosis than thus far seen in the literature. Osteopontin, tropomyosin and orosomucoid are only a couple of the molecules investigated using MALDI-MSI in ovarian cancer research. This study, in line with the available literature, proves the potential of MALDI-MSI to overcome the current limitations of classic histopathological examination giving a more in-depth insight into the tissue structure and thus lead to the more accurate differential diagnosis of ovarian tumors, especially in the most challenging cases.

137Cs γ Ray and 28Si Irradiation Induced Murine Hepatocellular Carcinoma Lipid Changes in Liver Assessed by MALDI-MSI Combined with Spatial Shrunken Centroid Clustering Algorithm: A Pilot Study.

Characterization of lipids by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is of great interest because not only are lipids important structural molecules in both the cell and internal organelle membranes, but they are also important signaling molecules. MALDI-MSI combined with spatial image segmentation has been previously used to identify tumor heterogeneities within tissues with distinct anatomical regions such as the brain. However, there has been no systematic study utilizing MALDI-MSI combined with spatial image segmentation to assess the tumor microenvironment in the liver. Here, we present that image segmentation can be used to evaluate the tumor microenvironment in the liver. In particular, to better understand the molecular mechanisms of irradiation-induced hepatic carcinogenesis, we used MALDI-MSI in the negative ion mode to identify lipid changes 12 months post exposure to low dose 28Si and 137Cs γ ray irradiation. We report here the changes in the lipid profiles of male C3H/HeNCrl mice liver tissues after exposure to irradiation and analyzed using the spatial shrunken centroid clustering algorithm. These findings provide valuable information as astronauts will be exposed to high-charge high-energy (HZE) particles and low-energy γ-ray irradiation during deep space travel. Even at low doses, exposure to these irradiations can lead to cancer. Previous studies infer that irradiation of mice with low-dose HZE particles induces oxidative damage and microenvironmental changes that are thought to play roles in the pathophysiology of hepatocellular carcinoma.

Electrospun α-Lactalbumin Nanofibers for Site-Specific and Fast-Onset Delivery of Nicotine in the Oral Cavity: An In Vitro, Ex Vivo, and Tissue Spatial Distribution Study.

Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette smoking, this being a shortcoming of the therapy. Here, we demonstrate that α-lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT applications. In vitro, nicotine-loaded nanofibers showed fast disintegration in water, with a weight loss up to 40% within minutes, and a faster nicotine release (26.1 ± 4.6% after 1 min of incubation) of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable nicotine dose (i.e., 7.9 ± 5.1 and 2.2 ± 0.3% nicotine was released from a lozenge and a sublingual tablet, respectively). Model-fitting of the release data indicated that the release mechanism of nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more efficient permeation of the nicotine released from the nanofibers [flux of 1.06 ± 0.22 nmol/(cm2·min)] compared to when dosing even a ten-fold concentrated nicotine solution [flux of 0.17 ± 0.14 nmol/(cm2·min)]. Moreover, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MS) imaging of ex vivo porcine buccal mucosa exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing, and biocompatible delivery system for nicotine, which can overcome the limitations of the currently marketed NRTs.

Novel Green Silver Nanoparticles as Matrix in the Detection of Small Molecules Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)

The number of studies utilizing the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has significantly increased over the past years. This technique features excellent sensitivity and speed of analysis for ionic compounds such as proteins, peptides, nucleic acids and small metabolites. Detection of small metabolites by MALDI-MS is essential for early diagnosis and monitoring of various diseases and understanding their pathogenesis. For metabolic analysis, conventional matrix and designed materials have certain limitations in terms of undesired aggregations, synthetic chemicals, low reproducibility and high cost. Herein, we report a novel application for enhanced MALDI-MS detection of small metabolites (MW < 500 Da) using biosynthesized silver nanoparticles (Ag-NPs) from the leaf parts of Cudrania tricuspidata as a green metal matrix. We were able to detect standard molecules (glucose, lysine, sucrose and glutamic acid) at a low detection limit (4–20 nmol) and achieve MALDI-MS profiling of these molecules at different dosages utilizing green metal matrix. The subsequent peaks were of higher intensity and with low background noise in comparison with those obtained using conventional α-cyano-4-hydroxycinnamic acid (CHCA) matrix and other nanomaterials. The tolerance assessment further demonstrated the analytical sensitivity of the green metal matrix in complex protein/salt mixture. In addition, we were able to detect 13 low molecular weight metabolites in 10× delude human healthy serum samples and another distinct 18 low molecular weight compounds in pancreatic cancer serum samples. Furthermore, the commonly used matrix, CHCA and other nanomaterial matrices, including silver, gold, SiO2@Ag and TiO2 nanoparticles, were also used in this study to compare with green noble metals (Ag) for the detection of small metabolites. Our work highlights the enhancement of green substrates as a matrix in the real-case analysis of small metabolites, besides the successful detection of the standard molecules using MALDI-MS. The green metal matrix offers future prospects in systems biology and disease detection and will have an industrial application for being economic, eco-friendly and rapid over the large scale chemical synthesis matrix.

Microanalytical techniques for phenotyping secondary xylem

Abstract The products of secondary xylem are of significant biological and commercial importance, and as a result, the biology of secondary growth and how intrinsic and extrinsic factors influence this process have been the subject of intense investigation. Studies into secondary xylem range in scale from the cellular to the forest stand level, with phenotypic analyses often involving the assessment of traits relating to cell morphology and cell wall chemical composition. While numerous techniques are currently available for phenotypic analyses of samples containing abundant amounts of secondary tissue, only a few of them (microanalytical techniques) are suitable when working with limiting amounts of secondary tissue or where a fine-scale resolution of morphological features or cell wall chemical composition is required. While polarised light microscopy, scanning electron microscopy, field emission-scanning electron microscopy and X-ray scattering and micro-tomography techniques serve as the most frequently used microanalytical techniques in morphotyping, techniques such as scanning ultraviolet microspectrophotometry, X-ray photoelectron spectroscopy, gas chromatography, Fourier-transform infrared spectroscopy and matrix-assisted laser desorption ionisation mass spectrometry serve as the most commonly used microanalytical techniques in chemotyping. Light microscopy, fluorescence microscopy, confocal laser scanning microscopy, transmission electron microscopy and Raman spectroscopy serve as dual micro morphotyping and chemotyping techniques. In this review, we summarise and discuss these techniques in the light of their applicability as microanalytical techniques to study secondary xylem.

Effect of matrices and additives on phosphorylated and ketodeoxyoctonic acid lipids A analysis by matrix-assisted laser desorption ionization-mass spectrometry.

Lipid A is a major compound of the outer membrane of gram-negative bacteria and is a key factor of bacterial virulence. As lipid A's structure differs among bacterial species and varies between strains of the same species, knowing its modifications is essential to understand its implications in the infectious process. To analyze these lipids, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) is a well-suited method that is fast and efficient. However, there are limitations with the matrix and additives used, such as the suppression of signal or prompt fragmentations that could give a false overview of lipid A composition in biological samples. For a comprehensive analysis of the entire lipid A species present in a sample, we tested 16 matrices and 11 additives on two commercial lipids A. The first commercial one contains single phosphorylation group, and the second contains two phosphorylation and two ketodeoxyoctonic acid (KDO) groups. The lipid A containing KDO groups was essentially detected by the 3-hydroxypicolinic acid (3-HPA) matrix, whereas the monophosphorylated lipid A could be detected by 13 matrices out of the 16. We also demonstrated that the signal of diphosphorylated lipid A can be enhanced with the use of additives in the matrix. Our study indicated that the best conditions to obtain a clear signal of both lipids A without prompt fragmentation was the use of 3-HPA with 10mM trifluoroacetic acid (TFA).

Spatial-temporal profiling of antibiotic metabolites using graphite dots-assisted laser desorption ionization mass spectrometry

Appropriate prescription of antibiotics requires the pharmacokinetic knowledge of the drugs and their metabolites in blood, and their distribution/retention in organ tissues. Here we report that highly crystalline graphite dots (GDs) allow for quantitative profiling of antibiotic metabolites in a spatial-temporal manner, in combination with matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI). GDs matrix features an ultra-clean background base line and high efficiency in ionization of small molecules, thus enabling quantification of sulfamethoxazole (SMZ) and its metabolites with limit of detection (LOD) in the femtomole range. Distinctly different from the other MS techniques, our approach is tolerant to high levels of salt contaminants in the complexed biological samples, thus minimizing the sample purification requirements and allowing for tests in small volumes. We have demonstrated quantitative measurements of the dynamic concentration changes of SMZ (m/z 276.27) and two metabolites, N4-hydroxy-SMZ (m/z 292.28) and N4-acetyl-SMZ (m/z 318.31) with only 1 μL mouse blood sample for each test. High-resolution distribution patterns of SMZ metabolites have directly been visualized a on the liver subsegments. Therefore, it allows for simultaneously acquisition of pharmacokinetic data in the blood combined with detailed hepatic zonation of SMZ metabolites for the first time. As a rapid, high-throughput platform to monitor small molecules in vivo, our approach of GDs-assisted MADLI MSI will foster the medical research on the antibiotic usage and drug development.

The structure of the Drosophila melanogaster sex peptide: Identification of hydroxylated isoleucine and a strain variation in the pattern of amino acid hydroxylation

In Drosophila melanogaster mating triggers profound changes in the behaviour and reproductive physiology of the female. Many of these post-mating effects are elicited by sex peptide (SP), a 36-mer pheromone made in the male accessory gland and passed to the female in the seminal fluid. The peptide comprises several structurally and functionally distinct domains, one of which consists of five 4-hydroxyprolines and induces a female immune response. The SP gene predicts an isoleucine (Ile14) sandwiched between two of the hydroxyprolines of the mature secreted peptide, but the identity of this residue was not established by peptide sequencing and amino acid analysis, presumably because of modification of the side chain. Here we have used matrix-assisted laser desorption ionisation mass spectrometry together with Fourier-transform ion cyclotron resonance mass spectrometry to show that Ile14 is modified by oxidation of the side chain - a very unusual post-translational modification. Mass spectrometric analysis of glands from different geographical populations of male D. melanogaster show that SP with six hydroxylated side chains is the most common form of the peptide, but that a sub-strain of Canton-S flies held at Leeds only has two or three hydroxylated prolines and an unmodified Ile14. The D. melanogaster genome has remarkably 17 putative hydroxylase genes that are strongly and almost exclusively expressed in the male accessory gland, suggesting that the gland is a powerhouse of protein oxidation. Strain variation in the pattern of sex peptide hydroxylation might be explained by differences in the expression of individual hydroxylase genes.

Visualisation of penetration of topical antifungal drug substances through mycosis-infected nails by matrix-assisted laser desorption ionisation mass spectrometry imaging.

Matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI) is a mass spectrometry-based technique, which can be applied for compound-specific imaging of pharmaceuticals in tissues samples. MALDI-MSI technology is widely used to visualise penetration and distribution profile through different tissues but has never been used with nail tissue. This study used MALDI-MSI technology to visualise distribution profile and penetration into ex vivo human mycosis-infected toenails of three antifungal active ingredients amorolfine, ciclopirox and naftifine contained in topical onychomycosis nail treatment preparations, marketed as Loceryl® , Ciclopoli® and Exoderil® . Three mycosis-infected toenails were used for each treatment condition. Six and twenty-four hours after one single topical application of antifungal drugs, excess of formulation was removed, nails were cryo-sectioned at a thickness of 20μm, and MALDI matrix was deposited on each nail slice. Penetration and distribution profile of amorolfine, ciclopirox and naftifine in the nails were analysed by MALDI-MSI. All antifungal actives have been visualised in the nail by MALDI-MSI. Ciclopirox and naftifine molecules showed a highly localised distribution in the uppermost layer of the nail plate. In comparison, amorolfine diffuses through the nail plate to the deep layers already 6hours after application and keeps diffusing towards the lowest nail layers within 24hours. This study shows for the first-time distribution and penetration of certain antifungal actives into human nails using MALDI-MSI analysis. The results showed a more homogeneous distribution of amorolfine to nail and a better penetration through the infected nails than ciclopirox and naftifine.

Mapping and Identification of Native Proteins of Developing Teeth in Mouse Mandibles.

Mass spectrometry imaging is a powerful tool of increasing utility due to its ability to spatially resolve molecular biomarkers directly from sectioned tissues. One hindrance to its universality is that no single protocol is sufficient for every tissue type, fixation, and pretreatment. Mineralized tissues are uniquely challenging as extensive decalcification protocols are necessary to achieve thin sections. In this study, we optimized a method to image tryptic peptides by matrix-assisted laser desorption ionization mass spectrometry of decalcified, formalin-fixed paraffin-embedded mouse hemimandibles. Using a combination of on-tissue MS/MS and hydrogel extraction LC-MS/MS, peptides from the enamel, dentin, periodontal ligament, alveolar bone, pulp, and other regions are identified and mapped. This breakthrough method provides a comprehensive approach to biomarker discovery in dental and craniofacial tissues which is highly relevant given that diseases originating from this region of the body are the most prevalent across all populations.

In situ label-free visualization of tissue distributions of salidroside in multiple mouse organs by MALDI-MS imaging

Detailed knowledge on the spatiotemporal distribution of drugs or drug candidates in bio-tissues is of vital importance for the determination of their underlying mechanisms of action as well as evaluation of potential side effects. The present work involved the establishment of a matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) method for the visualization of distribution of salidroside, a bioactive ingredient of Rhodiola L. Crassulaceae, in multiple mouse tissue sections. 1,5-Diaminonaphthalene (1,5-DAN) hydrochloride was selected as the most suitable matrix by comparing the signal intensity produced by MALDI-MS analysis following cocrystallization of salidroside with different matrix candidates. The matrix deposition procedure was systematically optimized to obtain the best sensitivity as well as homogeneous matrix coverage on the tissue sections. The measurements were carried out in the negative reflectron ion mode (m/z 300–350 Da) with a spatial resolution of 200 μm. The proposed method demonstrated good performance with respect to specificity, linearity, and sensitivity, and the method exhibited a relatively reasonable run time. The approach was applied to determine the temporal-spatial location of salidroside in the kidney, liver, spleen, heart, lung, and brain following intravenous administration of the drug in mice. The results indicate that salidroside exhibited a heterogeneous distribution in the kidney and heart, and it can be rapidly eliminated by kidney 5 min after administration. The distribution information obtained by the in situ and label-free imaging technique proposed herein may benefit to our understanding of the activities and potential transient toxicities of salidroside in various organs.

Ozonization of Tissue Sections for MALDI MS Imaging of Carbon-Carbon Double Bond Positional Isomers of Phospholipids.

Visualizing the differential distribution of carbon-carbon double bond (C═C db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of C═C db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 μm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.

Fluoresceinated Aminohexanol Tethered Inositol Hexakisphosphate: Studies on Arabidopsis thaliana and Drosophila melanogaster and Docking with 2P1M Receptor.

Inositol hexakisphosphate (InsP6; phytic acid) is considered as the second messenger and plays a very important role in plants, animals, and human beings. It is the principal storage form of phosphorus in many plant tissues, especially in dry fruits, bran, and seeds. The resulting anion is a colorless species that plays a critical role in nutrition and is believed to cure many diseases. A fluoresceinated aminohexanol tethered inositol hexakisphosphate (III) had been synthesized earlier involving many complicated steps. We describe here a simple two-step synthesis of (III) and its characterization using different techniques such as matrix-assisted laser desorption ionization mass spectrometry, tandem mass spectrometry, and Fourier transform infrared, ultraviolet–visible, ultraviolet-fluorescence, 1H nuclear magnetic resonance (NMR), and two-dimensional NMR spectroscopies. The effect of (III) has been investigated in the model systems, Arabidopsis thaliana and Drosophila melanogaster. Using Schrodinger software, computational studies on the binding of (III) with the protein 2P1M (Auxin-receptor TIR1-adaptor ASK1 complex) has revealed strong binding propensity with this compound. These studies on the fluoresceinated tethered phytic acid could have far reaching implications on its efficacy for human health and treatment of diseases (cancer/tumor and glioblastoma) and for understanding phosphorous recycling in the environment, especially for plant systems.

Lipid profiles in Hoffa’s fat pad as biomarker for cartilage regeneration and osteoarthritis development

Purpose: Early detection of knee osteoarthritis (OA) is critical for possible preventive treatment. Specific biomarkers for prognosis and early diagnosis of OA are lacking. Therefore, in this study, we analyzed the lipid profiles of different tissue types within Hoffa’s fat pad (HFP) of OA and cartilage defect (CD) patients, using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). The HFP has already been shown to play an important role in the inflammatory process in OA by prostaglandin release.

Discrimination of bovine milk from non-dairy milk by lipids fingerprinting using routine matrix-assisted laser desorption ionization mass spectrometry

An important sustainable development goal for any country is to ensure food security by producing a sufficient and safe food supply. This is the case for bovine milk where addition of non-dairy milks such as vegetables (e.g., soya or coconut) has become a common source of adulteration and fraud. Conventionally, gas chromatography techniques are used to detect key lipids (e.g., triacylglycerols) has an effective read-out of assessing milks origins and to detect foreign milks in bovine milks. However, such approach requires several sample preparation steps and a dedicated laboratory environment, precluding a high throughput process. To cope with this need, here, we aimed to develop a novel and simple method without organic solvent extractions for the detection of bovine and non-dairy milks based on lipids fingerprint by routine MALDI-TOF mass spectrometry (MS). The optimized method relies on the simple dilution of milks in water followed by MALDI-TOF MS analyses in the positive linear ion mode and using a matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (super-DHB) solubilized at 10 mg/mL in 70% ethanol. This sensitive, inexpensive, and rapid method has potential for use in food authenticity applications.

Capillary zone electrophoresis of bacterial extracellular vesicles: A proof of concept

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.

Unique and highly specific cyanogenic glycoside localization in stigmatic cells and pollen in the genus Lomatia (Proteaceae).

Floral chemical defence strategies remain understudied despite the significance of flowers to plant fitness, and the fact that many flowers contain secondary metabolites that confer resistance to herbivores. Optimal defence and apparency theories predict that the most apparent plant parts and/or those most important to fitness should be most defended. To test whether within-flower distributions of chemical defence are consistent with these theories we used cyanogenic glycosides (CNglycs), which are constitutive defence metabolites that deter herbivores by releasing hydrogen cyanide upon hydrolysis. We used cyanogenic florets of the genus Lomatia to investigate at what scale there may be strategic allocation of CNglycs in flowers, what their localization reveals about function, and whether levels of floral CNglycs differ between eight congeneric species across a climatic gradient. Within-flower distributions of CNglycs during development were quantified, CNglycs were identified and their localization was visualized in cryosectioned florets using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Florets of all congeneric species studied were cyanogenic, and concentrations differed between species. Within florets there was substantial variation in CNglyc concentrations, with extremely high concentrations (up to 14.6 mg CN g-1 d. wt) in pollen and loose, specialized surface cells on the pollen presenter, among the highest concentrations reported in plant tissues. Two tyrosine-derived CNglycs, the monoglycoside dhurrin and diglycoside proteacin, were identified. MALDI-MSI revealed their varying ratios in different floral tissues; proteacin was primarily localized to anthers and ovules, and dhurrin to specialized cells on the pollen presenter. The mix of transient specialized cells and pollen of L. fraxinifolia was ~11 % dhurrin and ~1.1 % proteacin by mass. Tissue-specific distributions of two CNglycs and substantial variation in their concentrations within florets suggests their allocation is under strong selection. Localized, high CNglyc concentrations in transient cells challenge the predictions of defence theories, and highlight the importance of fine-scale metabolite visualization, and the need for further investigation into the ecological and metabolic roles of CNglycs in floral tissues.

Charge Distribution between Different Classes of Glycerophospolipids in MALDI-MS Imaging.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is widely used to visualize and analyze the distribution of membrane lipids in an increasingly large number of applications. In this context, different lipoforms of glycerophospholipids (GPLs) are among the prime targets of interest. For this group of analytes, however, ion suppression effects have been described to strongly favor the detection of certain GPL classes over others, thereby hampering the analysis of suppressed species and greatly restraining quantitative analysis. These effects are generally attributed to the distribution of available charge carriers during the MALDI process. Here, we present a systematic investigation of charge distribution between different classes of GPL under MALDI-MSI conditions. For this, we constructed arrays of artificial tissues with different formulated lipid composition that contained predefined amounts of only two specific GPL classes and analyzed them with MALDI-MSI in positive- and negative-ion modes. Next to a characterization of expected ion suppression effects, analysis of these binary systems revealed yet undescribed signal intensity enhancement for the combinations of certain GPL classes. Furthermore, the comprehensive data allowed us to compile a hierarchy of charge affinities for the investigated GPL classes in both polarities. Additional experiments revealed that laser post-ionization (MALDI-2) has great potential to overwrite changes in signal intensity caused by charge distribution among different GPL classes observed in standard MALDI-MSI.