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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.

Abiotic and biotic stresses are the main reasons of substantial crop yield losses worldwide. Research devoted to reveal mechanisms of plant reactions during their interactions with the environment are conducted on the level of genome, transcriptome, proteome, and metabolome. Data obtained during these studies would permit to define biochemical and physiological mechanisms of plant resistance or susceptibility to affecting factors/stresses. Metabolomics based on mass spectrometric techniques is an important part of research conducted in the direction of breeding new varieties of crop plants tolerant to the affecting stresses and possessing good agronomical features. Studies of this kind are carried out on model, crop and resurrection plants. Metabolites profiling yields large sets of data and due to this fact numerous advanced statistical and bioinformatic methods permitting to obtain qualitative and quantitative evaluation of the results have been developed. Moreover, advanced integration of metabolomics data with these obtained on other omics levels: genome, transcriptome and proteome should be carried out. Such a holistic approach would bring us closer to understanding biochemical and physiological processes of the cell and whole plant interacting with the environment and further apply these observations in successful breeding of stress tolerant or resistant crop plants.

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 including flavonoid glycoconjugates. Resolution of the applied mass spectrometers plays an important role in structural studies of mixtures of the target compounds isolated from biological material. High-resolution analyzers allow obtaining information about elemental composition of the analyzed compounds. Application of various mass spectrometric techniques, including different systems of ionization, analysis of both positive and negative ions of flavonoids, fragmentation of the protonated/deprotonated molecules and in some cases addition of metal ions to the studied compounds before ionization and fragmentation, may improve structural characterization of natural products. In our review we present different strategies allowing structural characterization of positional isomers and isobaric compounds existing in class of flavonoid glycoconjugates and their derivatives, which are synthetized in plants and are important components of the human food and drugs as well as animal feed.

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.

Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome's complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.

International audience; Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arahidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase H PLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated.

The preconcentration of aqueous solutions containing atrazine degradation products was investigated using solid-phase extraction on octadecyl and cation-exchanger silica phases. The retention and elution steps were studied and evaluated separately in order to define and optimize the critical experimental parameters involved. A strategy which combines sequentially both phases is proposed to fractionate compounds into two groups of increasing polarities: firstly, the native pesticide, hydroxyatrazine and most chlorotriazines on octadecyl support, and secondly monodealkylated hydroxytriazines, ammeline and ammelide on cation-exchanger. This tandem procedure was successfully applied for analysing and quantifying atrazine ozonation products and its efficiency demonstrated using [U-ring 14C]-labelled atrazine experiments.

In contrast to the current view of kinetin (K, N(6)-furfuryladenine) as an unnatural and synthetic cytokinin, recently it has been identified in plant DNA and plant extract. Here we describe identification of K in human urine using chromatography/mass-spectrometry analysis for the first time. The amount of kinetin in urine taken from unhealthy patients lung carcinoma was established to be 0.5 ng in 20 ml and a 100-fold reduced amount in healthy subjects. Since this rare base is a potential source of structural constrains it has to be removed from DNA by enzymatic DNA-repair reactions. It seems that the presence of kinetin in human is linked to oxidative damage processes.

HPLC combined with electrospray ionization MS (LC–ESI-MS) was used for the identification of isoflavonoids and their glycosides in extracts from white lupin (Lupinus albus) tissues. The applicability of LC–ESI-MS for the simultaneous analysis of secondary metabolites of different polarities (free aglycones and their glycosides) is demonstrated. For optimization of the chromatographic and MS conditions, genistin (genistein 7-O-β-glucoside) and rutin [quercetin 3-O-β-(6″-O-α-rhamnosyl glucoside)] were used as standards. The detection limit for the analysis of genistin by HPLC was determined to be 20 ng (ca. 50 pmol). The analytical conditions established were employed to analyse isoflavonoids and their glycosidic conjugates obtained from lupin root extracts by solid phase extraction. Four glycosides and seven isoflavonoid aglycones were detected in the analysed samples. Copyright © 1999 John Wiley & Sons, Ltd.

The ability of liquid chromatography combined with positive-ion low-collision-energy tandem mass spectrometry for screening and characterization of atrazine metabolites in aqueous solutions is examined. The strategy used was defined after investigations on the structure and origin of the main daughter ions through experiments done by desorption chemical ionization with NH3 or ND3 as reagent gas. Following this, a three-step procedure was proposed which included parent-ion spectra based on [XCNC(NH)NH2]+ (with X Cl or OH) ions apparently formed via ion-dipole complexes and found to be highly diagnostic for chlorotriazines and hydroxytriazines respectively.

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.