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The aim of this study was to demonstrate the metabolic profile of post-culture medium as an expression of cell suspension metabolic activity of the tree fern Cyathea delgadii Sternb. The molecular profile of the tree fern’s cell culture has been never described, according to our knowledge. The cell suspension was established using ½ MS medium supplemented with various concentrations of 2,4-D and BAP. The optimal concentrations were 2.0 mg·L−1 and 0.2 mg·L−1, respectively. The cell suspension initially showed an organized system of cell division and later unorganized cell proliferation. LC-MS and GC-MS were used to identify the chemical composition of the post-culture medium. The LC-MS analysis results suggested that the color of liquid medium could be due to the presence of flavonoid derivatives, as this group of compounds was represented by eight compounds. After GC-MS analysis based on retention indexes and thanks to mass spectra comparison, 130 natural products were recognized, belonging to various classes of primary and secondary metabolites.

The aim of this study was to evaluate the utility of a hydroalcoholic extract of Hippocastani flos (HF) in wound healing, especially in regards to cell viability, proliferation, reactive oxygen species (ROS) generation and proinflammatory cytokine production using an endothelial cell line [human umbilical vein endothelial cell (HUVEC) line EAhy926]. Antibacterial and anti-hyaluronidase activity was also evaluated. The phytochemical profile was determined using an the UPLC–MS. The UPLC–MS analysis confirmed the presence of nine flavonoids, mainly kaempferol and quercetin derivatives. Specifically, the HF extract contained a high content of polyphenols (88.84 ± 4.13 mg chlorogenic acid equivalents/g dw), including flavonoids (15.68 ± 0.04 mg/quercetin equivalents/g dw). Antioxidant properties were demonstrated using FRAP assays (IC0.5 = 159.82 μg/mL). The HF extract exhibited a level of anti-hyaluronidase activity (IC50 = 0.86 mg/mL) similar to the positive control kaempferol (IC50 = 0.78 mg/mL). The antimicrobial properties of HF were shown against Staphylococcus aureus (MIC, MBC = 12.5 mg/mL). After exposing the endothelial cells to HF extract in vitro, it did not observed any change in the cell viability, proliferation, wound healing or proinflammatory cytokine production, but there was a dose dependent-reduction in ROS generation. In conclusion, HF extract exhibits anti-hyaluronidase activity, antimicrobial properties, and reduces ROS production. These observations may partly justify the use of HF in supporting wound healing.

Dimers of phenylpropanoid acids play an important role in cross-linking of polysaccharide chains present in the cell wall. These compounds are responsible not only for mechanical properties of cell walls, but also for plant tolerance to both biotic and abiotic stresses. Due to conjugated double bonds system these molecules are efficient radicals scavengers and UV light shield. Moreover, the presence of these natural products in many plant-derived foods provides a variety of health benefits and lowers disease risk. Therefore development of efficient methods of phenylpropanoid cyclobutane dimers and dehydrodimers analysis is of the highest interest. Liquid chromatography coupled to mass spectrometry with electrospray ionization is an analytical technique of choice for identification and – within certain limits – structural characterization of low molecular weight secondary metabolites in samples of plant origin. Utilization of high resolution analyzers in mass spectrometers enables accurate mass measurements, which allows to determine the elemental composition of these compounds with at least 5 ppm accuracy. The ions produced with electrospray ionization have low internal energy and their fragmentation can be generated by collision induced dissociation (CID MS/MS). LC/MS/MS system was used to profile phenylpropanoid dimers in barley leave cell wall extracts. Fifteen natural products were recognized after structural analysis of recorded CID MS/MS spectra, some of them were isomeric. We were able to detect: four isomers of two phenylpropanoid acids, three cyclobutane ferulic acid dimers, six isomeric ferulic acid dehydrodimers, one mixed dehydrodimer of sinapic and coumaric acid and one decarboxylated dehydrodimer of ferulic acid. Differentiation of isomeric compounds belonging to both classes of dimers, and their further quantitative analysis, will have influence on research directed to biological role of the dimers in plant responses to environmental biotic and abiotic factors.

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material. Plant tissues contain thousands of natural products fulfilling different roles in plant physiology and biochemistry. These natural products have various biological activities in respect to plants synthesizing them, in their responses to different environmental stresses and are also active principles of food supplements and pharmaceuticals of plant origin. Flavonoids constitute a large group of phenolic secondary metabolites and are probably produced by all terrestrial plant species. More than 9000 glycoconjugates of flavonoids are presently known in the plant kingdom and more than 50 of them may be present in a single plant. For this reason methods of identification and analysis of this group of compounds are particularly demanded. Due to a high number of metabolites present in plant extracts, the isolation and purification of most compounds in amounts suitable for unambiguous characterization with NMR methods is often impossible. For these reasons elaboration of strategies for sufficiently precise structural characterization of compounds present in mixture samples is currently a primary task. Mass spectrometry, thanks to application of different physical phenomena for ionization, separation and detection of analyzed molecules, became the method of choice among analytical methods applied for identification, structural characterization and quantitative analysis of the natural products. Methods of analysis of differently substituted flavonoids (O- and C-glycosides, differentiation of various oligosaccharidic substituents, detection of acylated compounds) are presented in the paper. A proper application of mass spectrometric methods in well-defined and strictly controlled technical parameters of analysis permits obtaining important structural information. Among others, recording collision induced dissociation mass spectra allows identification of compounds after comparison of the registered MS spectra with these present in the existing databases.

Liquid chromatography coupled to mass spectrometry (MS) with electrospray ionization (ESI) is one of analytical techniques to obtain accurate results of low molecular weight aromatic compounds in biological samples of different origin. The interpretations of mass spectra of these aromatic compounds in the negative spectra registered in the full scan MS mode may be uneasy due to presence of deprotonated molecules [M-H](-) from different co-eluting entities, fragment ions created after the break-up of precursor ions and also ions representing modified molecules clusters. Thus, the first aim of this study was to evaluate general parameters during analysis performed in the full scan MS or MS/MS mode. Secondly, to set general fragmentation rules for aromatic compounds and entities in a complex biological matrix. We established that different groups of low molecular weight phenolic acids form unique adduct ions and additionally registration LC/MS/MS spectra with two different collision energies may allow for differentiating isomeric or isobaric molecules. These findings together with some general fragmentation rules can facilitate identifications of aromatic acids as we outlined in the sample of cold-pressed rose-hip oil and lupine leaves extract.

Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznan, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznan.

Plant responses to abiotic stresses are very complex phenomena with individual characteristics for various species. Abiotic stresses (e.g. drought, salinity, flooding, cold, heat, UV radiation, heavy metals, etc.) strongly affect plant growth and development. It is estimated that they are the cause of more than 50 % of crop yield losses. Abiotic stresses are known to activate a multigene response resulting in the changes in various proteins and primary and secondary metabolite accumulation. Therefore, proteomic and metabolomic approaches are becoming very important and powerful tools used in studying plants’ reaction to various stimuli. Precise analysis of proteome and metabolome is essential for understanding the fundamentals of stress physiology and biochemistry. In this review, we focus on recent reports concerned to the influence of abiotic stresses on changes in the level of different protein groups and metabolite classes. Basic information about physicochemical methods applied to qualitative and quantitative analyses of biopolymers and natural products is also briefly presented.

Flavonoid glycoconjugates from roots and leaves of eight North America lupine species (Lupinus elegans, Lupinus exaltatus, Lupinus hintonii, Lupinus mexicanus, Lupinus montanus, Lupinus rotundiflorus, Lupinus stipulatus, Lupinus sp.), three Mediterranean species (Lupinus albus, Lupinus angustifolius, Lupinus luteus) and one species from South America domesticated in Europe (Lupinus mutabilis) were analyzed using two LC/MS systems: low-resolution ion trap instrument and high-resolution quadrupole-time-of-flight spectrometer. As a result of the LC/MS profiling using the CID/MS(n) experiments structures of 175 flavonoid glycoconjugates found in 12 lupine species were identified at three confidence levels according to the Metabolomic Standard Initiative, mainly at level 2 and 3, some of them were classified to the level 1. Among the flavonoid derivatives recognized in the plant extracts were isomeric or isobaric compounds, differing in the degree of hydroxylation of the aglycones and the presence of glycosidic, acyl or alkyl groups in the molecules. The elemental composition of the glycoconjugate molecules was established from the exact m/z values of the protonated/deprotonated molecules ([M+H](+)/[M-H](-)) measured with the accuracy better than 5 ppm. Information concerning structures of the aglycones, the type of sugar moieties (hexose, deoxyhexose or pentose) and, in some cases, their placement on the aglycones as well as the acyl substituents of the flavonoid glycoconjugates was achieved in experiments, in which collision-induced dissociation was applied. Flavonoid aglycones present in the studied O-glycoconjugates were unambiguously identified after the comparison of the pseudo-MS(3) spectra with the spectra registered for the standards. Isomers of flavonoid glycoconjugates, in which one or two sugar moieties were attached to 4'- or 7-hydroxyl groups or directly to the C-6 or C-8 of the aglycones, could be distinguished on the basis of the MS(2) spectra. However, the collision energy applied in the CID experiments had to be optimized for each group of the compounds and there were no universal settings that allowed the acquisition of structural information for all the compounds present in the sample. Information obtained from the flavonoid conjugate profiling was used for the chemotaxonomic comparison of the studied lupine species. A clear-cut discrimination of the Mediterranean and North American lupines was obtained as a result of this analysis.

The major cause of mortality in patients with chronic kidney disease (CKD) is atherosclerosis related to traditional and non-traditional risk factors. However, the understanding of the molecular specificity that distinguishes the risk factors for classical cardiovascular disease (CVD) and CKD-related atherosclerosis (CKD-A) is far from complete. In this study we investigated the disease-related differences in the proteomes of patients with atherosclerosis related and non-related to CKD. Plasma collected from patients in various stages of CKD, CVD patients without symptoms of kidney dysfunction, and healthy volunteers (HVs), were analyzed by a coupled label-free and mass spectrometry approach. Dysregulated proteins were confirmed by an enzyme-linked immunosorbent assay (ELISA). All proteomic data were correlated with kidney disease development and were subjected to bioinformatics analysis. One hundred sixty-two differentially expressed proteins were identified. By directly comparing the plasma proteomes from HVs, CKD, and CVD patients in one study, we demonstrated that proteins involved in inflammation, blood coagulation, oxidative stress, vascular damage, and calcification process exhibited greater alterations in patients with atherosclerosis related with CKD. These data indicate that the above nontraditional risk factors are strongly specific for CKD-A and appear to be less essential for the development of “classical” CVD.

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.

Little biochemical information is available regarding the responses of embryo axes to Fusarium oxysporum during the stages of seed germination and early seedling growth of yellow lupine, i.e. in the heterotrophic phase and during the transition from the heterotrophic to the autotrophic phase [1] . In the experimental protocol used in this study germinated yellow lupine seeds, both non-inoculated and inoculated with F. oxysporum , were cultured for 96 h in perlite. An analysis of soluble carbohydrates, between 48 and 96 h of culture, shows a decrease in sugar content, i.e. that of sucrose and fructose in infected embryo axes of germinated seeds and that of sucrose and glucose in the controls at 72 h. After infection, lower levels of sucrose, glucose and fructose were observed in infected axes than in controls. Invertase activity (EC 3.2.1.26) in infected tissues was higher than in the controls with the exception of acid invertase activity at 96 h. The highest post-infection alkaline and acid invertase activity was recorded in 48 h embryo axes of germinated seeds. An HPLC analysis in the period between 48 and 96 h after infection showed a decrease in the level of glycosylated isoflavonoids, i.e. genistein 7-O-glucoside, 2′OH genistein 7-O-glucoside, genistein 4′,7-O-diglucoside, 2′OH genistein malonyl glucoside and 2′OH genistein malonyl glucoside, which was particularly marked at 72 h after infection. Concentrations of these glucosides in 72 h and 96 h infected tissues were lower than in controls. We need to stress here a very high isoflavonoid content observed in intact embryo axes of germinating seeds at 48 h after inoculation, i.e. shortly after the embryo axes penetrated the seed coat. At this time point in infected tissues the concentrations of genistein glucosides and that of a free aglycone, genistein, were many times higher than in the controls. At later time points after inoculation genistein content was also higher than in the controls. A different tendency to that observed in infected axes was seen in the control specimens, where the level of flavonoid glucosides rose in the period between 48 and 72 h of culture, followed by a subsequent decrease. The activity of phenylalanine ammonialyase (PAL, EC. 4.3.1.5) in infected embryo axes of germinating seeds was higher than in the controls and the highest post-infection activity of this enzyme was observed in 48 h axes. At this time point, a considerable post-infection accumulation was observed using semi-quantitative RT-PCR for transcripts of phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS), which was consistent with the high level of isoflavonoids. These results indicate that in yellow lupine during the stages of seed germination and early seedling growth changes in carbohydrate and isoflavonoid metabolism occur in embryo axes in response to infection with F. oxysporum f.sp. lupini . These changes may be included in the active protection mechanism in these axes.

Introduction – Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono- or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. Methodology – Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high-performance liquid chromatography combined with high-resolution tandem (quadrupole-time of flight, QToF) mass spectrometry. Results – The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p-coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high-resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. Perspectives – The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species. Copyright © 2009 John Wiley & Sons, Ltd.

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzyme's active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.

Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H](+), sodiated [M + Na](+) or deprotonated [M - H](-) molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na](+) ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H](+) and [M - H](-) ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides.

In wheat (Triticum aestivum L.), treatment with herbicide safeners enhances the expression of enzymes involved in pesticide detoxification and reduces crop sensitivity to herbicides. Since these same enzymes are involved in plant secondary metabolism, it was of interest to determine whether or not the safener cloquintocet mexyl perturbed phenolic metabolism in wheat seedlings. LC/ESI/MS analysis identified 14 phenolic substrates in the shoots of young wheat plants. Fragmentation imposed by collision induced dissociation identified specific C-glycosidic conjugates of 4',5,7-trihydroxflavone (apigenin), 3',4',5,7-tetrahydroxyflavone (luteolin) and 3'-O-methylluteolin. Treatment of 7-day-old wheat shoots with cloquintocet mexyl resulted in an accelerated depletion of the conjugates of all three flavones, most notably with the glycosides of luteolin. In contrast, safener treatment caused the selective accumulation of 4',5,7-trihydroxy-3',5'-dimethoxyflavone (tricin) and the phenylpropanoid ferulic acid. Changes in phenolic content were associated with an increase in O-methyltransferase and C-glucosyltransferase activity toward flavonoid substrates as well as the classic enhancement of detoxifying glutathione transferases. Our results suggest that in addition to altering the capacity of wheat to metabolise herbicides and other xenobiotics, safeners can also cause a selective shift in the metabolism of endogenous phenolics.

Extracts from plantlets of different species of the genus Drosera, grown as in vitro cultures, were evaluated for the level of phenolic secondary metabolites from the group of naphthoquinones and flavonols. The profiles of natural products in the extracts obtained from different species were monitored by HPLC with UV detection at 260 and 330 nm. On the basis of the data obtained, Drosera binata, the species with the highest amount of plumbagin, was selected for further studies. The most effective method of extraction of quinones was established and the composition of phenolic secondary metabolites in the tissues was determined. For the identification of phenolic compounds, HPLC-UV and HPLC-ESI/MS were applied. Copyright © 2005 John Wiley & Sons, Ltd.

Deciphering of the plant metabolome is one of the most difficult analytical tasks in functional genomic research. Studies directed at the gene or protein expression are well established, sequencing analyses of these kinds of biopolymers on genome or proteome level are possible. This is not the case for metabolites, where identification in single sample of many chemical entities of different elemental composition and structures and various physicochemical properties is necessary. Different instrumental methods are applied for identification of metabolites but none of them allows obtaining unambiguous structural information about more than 500 compounds in single mixture (metabolite profiling). This is a much smaller number of metabolites than is predicted for single plant metabolome. However, instrumental approaches were proposed (metabolite fingerprinting) in which biochemical phenotype of an organism may be estimated, but identification of individual compounds is not possible.

A mass spectrometric method based on the combined use of electrospray ionization, collision-induced dissociation and tandem mass spectrometry at high mass resolution has been applied to an investigation of the structural characterization of genistein-7-O-β- d -glucoside (5,7,4′-trihydroxyisoflavone). The product ion mass spectrum of [M−H]− ions shows neutral losses of the glycan residue (162 Da) and of the glycan residue + H (163 Da) by rearrangement and scission, respectively, where the latter loss dominates at higher collision energies. The genistein moiety remained intact and only minor fragmentation of the glucose moiety was observed. The low-energy product ion mass spectrum of [M+H]+ ions shows extensive fragmentation of the glucose moiety, though at low ion signal intensity, loss of the glycan residue, and simple fragmentation of the genistein moiety that permits characterization of the substituents in the A and B rings. The use of elevated cone voltages permitted observation of product ion mass spectra of selected primary fragment ions. Product ion mass spectra examined at high mass resolution allowed unambiguous determination of the elemental composition of fragment ions. Fragmentation mechanisms and ion structures have been proposed.

Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except alpha-chaconine and alpha-solanine, in the extracts from dried tuber skin alpha-solamargine and alpha-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized.

Mass spectrometric fragmentation patterns of herbacetin 3-O-glucopyranoside-8-O-glucuronopyranoside (1), gossypetin 3-O-glucopyranoside-8-O-glucuronopyranoside (2) and takakin 7-O-glucopyranoside-8-O-glucuronopyranoside (3) were elucidated from mass spectra obtained with electrospray ionisation. The usefulness of the fragmentation patterns observed in the positive and negative mode for structural elucidation of the studied compounds is discussed. The fragmentation of 3 was substantially different from 1 and 2, especially in the negative mode. In order to explain these differences, theoretical calculations were performed. Copyright © 2003 John Wiley & Sons, Ltd.

In this paper we show that transfer ribonucleic acids (tRNAs) catalyze the Diels-Alder cycloaddition reaction. A new DNA oxidative damage product, 6-furfuryladenine (kinetin) or its riboside (diene), was transformed with dimethyl acetylenedicarboxylate or maleic anhydride (dienophile). The reaction proceeds in the presence of tRNA at high pressure but not at ambient condition. If so tRNA in prebiotic conditions (RNA world) had at least two functions: catalytic and a carrier of genetic information. It means that tRNA at high pressure shows catalytic properties and is a true Diels-Alderase.

Two flavonoid sulphates, i.e. quercetin 3-O-sulphate-7-O-α-arabinopyranoside and kaempferol 3-O-sulphate-7-O-α-arabinopyranoside, were isolated from leaves of Atriplex hortensis L. The structures of these compounds were established by UV, 1H and 13C NMR, 2D NMR and MS spectra. The compounds were isolated for the first time from plant material.

The aim of this study was to characterize the thermal stability of diclofenac sodium both alone and in the inclusion complex with β-cyclodextrin in the solid state, by determination of the number of the products of its decomposition, which were identified by GC-MS. The molar ratio of diclofenac sodium in the inclusion complex with β-cyclodextrin was 1:1. The decomposition of diclofenac sodium both alone and in inclusion complex with β-cyclodextrin occurred according to the first-order reaction. The HPLC of the samples thermostated at 80°C gave five products of decomposition, which were identified by GC-MS. Diclofenac sodium in the inclusion complex with β-cyclodextrin was more thermally stable. Thermal decomposition of diclofenac sodium leads to formation of five products, of which 4-chloro-10H-9-acridinone had not been reported previously in the literature.

Various mass spectrometric techniques were used for the structural elucidation of flavonoid glycosides isolated from green parts of yellow lupin plants (Lupinus luteus). A methodological approach is proposed consisting of (1) extraction and purification of flavonoid glycosides from plant tissues, (2) registration of liquid secondary ion mass spectra of intact compounds and (3) gas chromatographic/mass spectrometric analyses of those compounds subjected to several types of chemical modification. On the basis of these data, it was possible to define the molecular mass of the glycosides, the structure of the aglycones, the configuration of sugar moieties and the position of glycosidic linkages between sugars in diglycosides or between aglycone and sugar moieties. Analysis of the mass spectrometric data permitted structural identification of four flavonoid diglycosides, where the aglycones had flavone, isoflavone and flavonol structures. They were recognized as: apigenin 7-O-(2″-O -rhamnopyranosyl)glucopyranoside (1), genistein 4′,7-O-diglucopyranoside (2), kaempferol 3-O -(6″-rhamnopyranosyl)glucopyranoside (3) and 3′- or 4′-methylquercetin 3-O-(6″-O -rhamnopyranosyl)glucopyranoside (4). Complete structural evaluation of flavonoid glycosides was possible with only submilligram quantities of samples needed to register various mass spectra. It was not possible, however, to define the anomeric configuration of the C-1 carbons in investigated glycosides. Copyright © 1999 John Wiley & Sons, Ltd.

The study aimed to detect features of human serum proteome that were associated with exposure to ionizing radiation. The analyzed group consisted of 46 patients treated with radical radiotherapy for larynx cancer; patients were irradiated with total doses in a range from 51 to 72 Gy. Three consecutive blood samples were collected from each patient: before the start, 2 weeks after the start, and 4-6 weeks after the end of radiotherapy. The low-molecular-weight fraction of the serum proteome (2,000-13,000 Da) was analyzed by the MALDI-ToF mass spectrometry. Proteome profiles of serum samples collected before the start of radiotherapy and during the early stage of the treatment were similar. In marked contrast, mass profiles of serum samples collected several weeks after the end of the treatment revealed clear changes. We found that 41 out of 312 registered peptide ions changed their abundance significantly when serum samples collected after the final irradiation were compared with samples collected at the two earlier time points. We also found that abundances of certain serum peptides were associated with total doses of radiation received by patients. The results of this pilot study indicate that features of serum proteome analyzed by mass spectrometry have potential applicability as a retrospective marker of exposure to ionizing radiation.

Background Mass spectrometric analysis of the blood proteome is an emerging method of clinical proteomics. The approach exploiting multi-protein/peptide sets (fingerprints) detected by mass spectrometry that reflect overall features of a specimen's proteome, termed proteome pattern analysis, have been already shown in several studies to have applicability in cancer diagnostics. We aimed to identify serum proteome patterns specific for early stage breast cancer patients using MALDI-ToF mass spectrometry. Methods Blood samples were collected before the start of therapy in a group of 92 patients diagnosed at stages I and II of the disease, and in a group of age-matched healthy controls (104 women). Serum specimens were purified and the low-molecular-weight proteome fraction was examined using MALDI-ToF mass spectrometry after removal of albumin and other high-molecular-weight serum proteins. Protein ions registered in a mass range between 2,000 and 10,000 Da were analyzed using a new bioinformatic tool created in our group, which included modeling spectra as a sum of Gaussian bell-shaped curves. Results We have identified features of serum proteome patterns that were significantly different between blood samples of healthy individuals and early stage breast cancer patients. The classifier built of three spectral components that differentiated controls and cancer patients had 83% sensitivity and 85% specificity. Spectral components (i.e., protein ions) that were the most frequent in such classifiers had approximate m/z values of 2303, 2866 and 3579 Da (a biomarker built from these three components showed 88% sensitivity and 78% specificity). Of note, we did not find a significant correlation between features of serum proteome patterns and established prognostic or predictive factors like tumor size, nodal involvement, histopathological grade, estrogen and progesterone receptor expression. In addition, we observed a significantly (p = 0.0003) increased level of osteopontin in blood of the group of cancer patients studied (however, the plasma level of osteopontin classified cancer samples with 88% sensitivity but only 28% specificity). Conclusion MALDI-ToF spectrometry of serum has an obvious potential to differentiate samples between early breast cancer patients and healthy controls. Importantly, a classifier built on MS-based serum proteome patterns outperforms available protein biomarkers analyzed in blood by immunoassays.

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.

The electron impact induced mass fragmentations of 16 new 2- and 4-alkylthio-5-chloro- and 2- and 4-alkylthio-5-bromouracils are presented. The elucidation of the fragmentation pathways is described. The fragmentation routes have been confirmed by accurate mass measurements and metastable transitions. It has been found that the position of uracil ring thiation may be deduced on the basis of the mass spectral fragmentation patterns.

The electron impact induced fragmentation of a large number of 2- and 4-S-(2-naphthoxy)-polymethyienethiouracils is discussed in detail, with the aid of exact mass measurements and observation of metastable decompositions. The 2- and 4-thiopyrimidine structures can be easily distinguished by their mass spectra.

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H](+). Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS(3) experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0287-2) contains supplementary material, which is available to authorized users.

Plant interactions with environmental factors cause changes in the metabolism and regulation of biochemical and physiological processes. Plant defense against pathogenic microorganisms depends on an innate immunity system that is activated as a result of infection. There are two mechanisms of triggering this system: basal immunity activated as a result of a perception of microbe-associated molecular patterns through pattern recognition receptors situated on the cell surface and effector-triggered immunity (ETI). An induced biosynthesis of bioactive secondary metabolites, in particular phytoalexins, is one of the mechanisms of plant defense to fungal infection. Results of the study on narrow leaf lupin (Lupinus angustifolius L.) plants infected with the anthracnose fungus Colletotrichum lupini and treated with fungal phytotoxic metabolites are described in the paper. The C. lupini phytotoxins were isolated from liquid cultures, purified and partially characterized with physicochemical methods. Accumulation of secondary metabolites on leaf surface and within the tissues of plants either infected, treated with the fungal phytotoxin or submitted to both treatments was studied using GC-MS and LC-MS, respectively. Substantial differences in isoflavone aglycones and glycoconjugate profiles occurred in response to different ways of plant treatment.

Background The proteomics approach termed proteome pattern analysis has been shown previously to have potential in the detection and classification of breast cancer. Here we aimed to identify changes in serum proteome patterns related to therapy of breast cancer patients. Methods Blood samples were collected before the start of therapy, after the surgical resection of tumors and one year after the end of therapy in a group of 70 patients diagnosed at early stages of the disease. Patients were treated with surgery either independently (26) or in combination with neoadjuvant chemotherapy (5) or adjuvant radio/chemotherapy (39). The low-molecular-weight fraction of serum proteome was examined using MALDI-ToF mass spectrometry, and then changes in intensities of peptide ions registered in a mass range between 2,000 and 14,000 Da were identified and correlated with clinical data. Results We found that surgical resection of tumors did not have an immediate effect on the mass profiles of the serum proteome. On the other hand, significant long-term effects were observed in serum proteome patterns one year after the end of basic treatment (we found that about 20 peptides exhibited significant changes in their abundances). Moreover, the significant differences were found primarily in the subgroup of patients treated with adjuvant therapy, but not in the subgroup subjected only to surgery. This suggests that the observed changes reflect overall responses of the patients to the toxic effects of adjuvant radio/chemotherapy. In line with this hypothesis we detected two serum peptides (registered m/z values 2,184 and 5,403 Da) whose changes correlated significantly with the type of treatment employed (their abundances decreased after adjuvant therapy, but increased in patients treated only with surgery). On the other hand, no significant correlation was found between changes in the abundance of any spectral component or clinical features of patients, including staging and grading of tumors. Conclusions The study establishes a high potential of MALDI-ToF-based analyses for the detection of dynamic changes in the serum proteome related to therapy of breast cancer patients, which revealed the potential applicability of serum proteome patterns analyses in monitoring the toxicity of therapy.