Liquid Chromatography Coupled With Mass Spectrometry Research Articles

Excretion of non-metabolized insecticides in honeydew of striped pine scale

Honeydew production is a characteristic of soft scales and other hemipteran insects. Honeydew has the capacity to alter the ecology of predators and parasitoids because it is used as a food resource and can contain insecticidal proteins from hemipteran host plants. We examined honeydew excreted by the striped pine scale (Hemiptera: Coccidae), Toumeyella pini (King), after feeding on pine trees treated with systemic insecticides to determine whether they could eliminate insecticidal compounds in honeydew. Imidacloprid and spirotetramat were applied at labeled rates to soil or foliage. Water sensitive paper was used to measure honeydew production and liquid chromatography coupled to mass spectrometry (LC-MS) to analyze excreted insecticide concentrations. Foliar and soil applications of imidacloprid caused a 25-fold reduction honeydew produced by scales six days after treatment (DAT). In contrast, spirotetramat treatments did not affect honeydew production. Parent compounds of both insecticides were detected in honeydew. However, on imidacloprid treated plants, these compounds were detected at similar concentrations in honeydew collected at 4 DAT from soil and foliar treatments. Imidacloprid was only detected from soil treatments at 8 DAT. Similarly, the spirotetramat parent compound was found 4 DAT after soil and foliar treatments, but only at 8 DAT in foliar treatments. At this time the concentration of spirotetramat in honeydew was six-fold higher than at 4 DAT. We conclude that striped pine scales excrete insecticides in honeydew even when the toxicant greatly reduces honeydew production. Honeydew excretion is thus a mechanism of bioaccumulation and has the potential to harm honeydew-feeding organisms.

Influence of the Extraction Conditions on the Antifungal Properties of Walnut Green Husk Isolates

The green husk is one of the major wastes produced during the walnut (Juglans regia L.) cultivation. However, the results presented in this article may contribute to extension of their usefulness as new and safe plant protection products. The results confirmed the activity of the walnut green husk extracts against plant (Alternaria alternata, Rhizoctonia solani, Botrytis cinerea, Fusarium culmorum, Phytophthora cactorum) and bee (Ascosphaera apis) pathogenic fungi, and showed that the extraction conditions differentiate the properties of the obtained isolates. The increase in the isolation temperature leads to a significant reduction in the antifungal activity of the extracts. The change of the extractant type modifies the resistance of individual fungi. The observed variations in the properties are consistent with the change of juglone content in these extracts. However, considering the juglone concentration provided 50% inhibition of the growth of individual mycelia, the observed differentiation of the antifungal properties of extracts should not be associated only with the juglone content. This observation, supported by the results obtained using gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled to mass spectrometry (LC-MS), suggests a synergistic effect of the other components on the antifungal activity of walnut green husk extracts.

The condensed tannins of Okoume (Aucoumea klaineana Pierre): A molecular structure and thermal stability study

In order to promote convenient strategies for the valorization of Aucoumea klaineana Pierre (Okoume) plywood and sawmill wastes industry in the fields of adhesives and composites, the total phenolic content of Okoume bark, sapwood and heartwood was measured. The molecular structure of tannins extracted from the bark was determined by Matrix Assisted Laser Desorption/Ionization Time-Of-Flight (Maldi-ToF) mass spectrometry and Fourier transform infrared spectroscopy (FTIR). The total phenolic content displayed significant difference (p = 0.001) between the bark, sapwood and heartwood which decreased as follows: 6 ± 0.4, 2 ± 0.8 and 0.7 ± 0.1% respectively. The pro-anthocyanidins content was also significantly different (p = 0.01) among the three wood wastes, and the bark was the richest in condensed tannins (4.2 ± 0.4%) compared to the sapwood (0.5 ± 0.1%) and heartwood (0.2 ± 0.2%). Liquid chromatography coupled mass spectroscopy (LC-MS) and Maldi-ToF analysis of the bark showed for the first time that Okoume condensed tannins are fisetinidin, gallocatechin and trihydroxyflavan based monomers and complex polymers obtained with glycosylated units. No free catechin or robitinidin units were detected, whereas distinctive dihydroxy or trihydroxyflavan-3-benzoate dimers were observed in the investigated condensed tannin extracts. FTIR analysis showed the occurrence of glucan- and mannan-like sugars in the condensed tannins, and Maldi-ToF highlighted that these sugars should account for ten glycosylated units chemically bonded with two fisetinidins and one gallocatechin trimer. The condensation of these polyphenols with formaldehyde led to Stiasny numbers of 83.3, 73.3 and 53.3% for the bark, sapwood and heartwood, respectively.

Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry.

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment.

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cutoff (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP with low molecular weight (11 kDa and 17 kDa) at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

Insight about cell wall remodulation triggered by rifampicin in Mycobacterium tuberculosis

Rifampicin plays an important role during the treatment of tuberculosis, which makes it to be recommended throughout the regimen. The molecular target for rifampicin activity and resistance is the bacterial RNA polymerase coded by rpoB. However, it has been observed that Mycobacterium tuberculosis could use different metabolic pathways contributing to drug activity/resistance. In this sense, Proteomics analysis has been a key aspect towards the understanding of the dynamic genome expression triggered by drugs and other M. tuberculosis hostile stimuli. Herein, we aimed to report the changes in the M. tuberculosis protein profile triggered by rifampicin. The M. tuberculosis H37Rv strain was submitted to 12, 24 and 48 h of rifampicin challenge, at the minimal inhibitory concentration (0.03 μg mL−1), and proteins were extracted. The protein identification was carried out by liquid chromatography coupled to mass spectrometry (LC-MS). Four proteins, Ino1 (Rv0046c), FabD (Rv2243), EsxK (Rv1197) and PPE60 (Rv3478) were statistically underexpressed over 48 h of rifampicin exposure, indicating that in addition to the known activity of rifampin in transcriptional machinery in M. tuberculosis, processes related to disturbance in cell wall synthesis and lipid metabolism in the bacillus are also triggered by rifampicin contributing to bacillus death.

Chemical Components and Hepatoprotective Mechanism of Xwak Granule in Mice Treated with Acute Alcohol.

Objective To evaluate the hepatoprotective mechanism of Xwak granule (Xwak) in treatment of mice with alcoholic liver injury via activating ERK/NF-κB and Nrf/HO-1 signaling pathways. Methods The chemical composition of Xwak was tested by liquid chromatography coupled with mass spectrometry (LC-MS). Herein, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical tests were performed in vitro. The hepatoprotective effect of Xwak was assessed at different concentrations (1.5, 3, and 6 g/kg) in a mouse model of alcoholic liver injury. Results Totally, 48 compounds, including 16 flavonoids, 8 tannins, 9 chlorogenic acids, and 15 other compounds, were identified from Xwak. Xwak showed to have a satisfactory antioxidant activity in vitro. In a group of Xwak-treated mice, the serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were decreased compared with a group of the mouse model of alcoholic liver injury. In addition, the levels of antioxidant enzymes, such as glutathione peroxidase (GSH-PX), total superoxide dismutase (T-SOD), and catalase (CAT), were noticeably increased and the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and interleukin-6 (IL-6) were markedly reduced in the liver of mice. The state of oxidative stress in the mouse model of alcoholic liver injury was improved after treatment with Xwak. The improvement of inflammation-mediated disruption may conducive to the Xwak activity in the control of liver injury. The signals of p-ERK1/2, p-NF-κB, COX-2, iNOS, CYP2E1, Nrf, and HO-1 were significantly induced in the liver of mice after treatment with Xwak. Conclusions The abovementioned findings indicated that the hepatoprotective mechanism of Xwak could be achieved by activating ERK/NF-κB and Nrf/HO-1 signaling pathways to alleviate oxidative stress and inflammatory.

Nano-liquid chromatography-mass spectrometry and recent applications in omics investigations.

Liquid chromatography coupled to mass spectrometry (LC-MS) is one of the most powerful tools in identifying and quantitating molecular species. Decreasing column diameter from the millimeter to micrometer scale is now a well-developed method which allows for sample limited analysis. Specific fabrication of capillary columns is required for proper implementation and optimization when working in the nanoflow regime. Coupling the capillary column to the mass spectrometer for electrospray ionization (ESI) requires reduction of the subsequent emitter tip. Reduction of column diameter to capillary scale can produce improved chromatographic efficiency and the reduction of emitter tip size increased sensitivity of the electrospray process. This improved sensitivity and ionization efficiency is valuable in analysis of precious biological samples where analytes vary in size, ion affinity, and concentration. In this review we will discuss common approaches and challenges in implementing nLC-MS methods and how the advantages can be leveraged to investigate a wide range of biomolecules.

A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry.

Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.

Plasma lipidomic biomarker analysis reveals distinct lipid changes in vascular dementia

Vascular dementia (VaD) is a complex neurocognitive disorder secondary to a variety of cerebrovascular lesions. Numerous studies have shown that lipid metabolism is involved in the pathobiology of the disease. We examined the plasma lipid profiles in VaD, with the expectation of identifying reliable lipid biomarkers for VaD. 49 VaD patients and 48 healthy controls were recruited from Bankstown-Lidcombe Hospital in Sydney, Australia.Lipids were extracted by single phase 1-butanol/methanol, and untargeted analysis was performed by liquid chromatography coupled-mass spectrometry (LC–MS/MS). Univariate analysis of variance was used to examine the differences in lipid classes and individual lipids between VaD and control groups. In an independent sample of 161 subjects from the Older Australian Twins Study (OATS), elastic net penalization for the generalized linear model (Glmnet) and Random Forest were applied to the lipid levels to subcategorise the sample into vascular cognitive impairment and controls. Most lipids belonging to the classes of ceramides (Cer), cholesterol esters (ChE) and phospholipids were significantly lower in VaD plasma, while glycerides were elevated compared to controls. Levels of ChE, Cer and the two lipid classes together achieved the best accuracy in discriminating VaD from controls, with more than 80% accuracy. The probable VaD group in the OATS sample predicted by the lipid levels showed greater impairment in most cognitive domains, especially attention and processing speed and executive function from controls but did not differ in white matter hyperintensities and DTI measures. As a conclusion, plasma lipids levels, in particular Cer and ChE, are abnormal in VaD and may help discriminate them from healthy controls. Understanding the basis of these differences may provide insights into the pathobiology of VaD.

Surfactin-producing Bacillus velezensis 1B-23 and Bacillus sp. 1D-12 protect tomato against bacterial canker caused by Clavibacter michiganensis subsp. michiganensis

Bacillus strains are known to produce cyclic lipopeptides that are capable of providing protection against plant pathogens. Such abilities could be utilized to protect greenhouse tomatoes against diseases including bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm). In the present study, Bacillus velezensis strain 1B-23 and Bacillus sp. strain 1D-12 were assessed for their potential biocontrol abilities against Cmm strain 98–1 (Cmm98–1). Both Bacillus strains interfered with growth of Cmm98–1 in vitro, as determined by agar plate assays to screen for microbial antagonism. Inoculation of Cmm98–1 infected tomato plants with B. velezenis 1B-23 or Bacillus sp. 1D-12 lead to significantly reduced disease incidence in a greenhouse setting. Liquid Chromatography coupled to Mass Spectrometry (LC-MS) of 1B-23 and 1D-12 extracts identified [Leu7]surfactin C13 (often called surfactin A), [Leu7]surfactin C14 (often called surfactin B) and [Leu7]surfactin C15 (often called surfactin C) in fractions of extracts that inhibited growth of Cmm98–1.

The enhanced photocatalytic performance toward carbamazepine by nitrogen-doped carbon dots decorated on BiOBr/CeO2: Mechanism insight and degradation pathways

Novel nitrogen-doped carbon dots (NCDs) modified BiOBr/CeO2 was constructed successfully via a facile method. The morphology, structure, optical and photo-electrochemical properties of the photocatalysts were characterized by various technologies. The photocatalytic performance was evaluated by the degradation of carbamazepine (CBZ) under simulated solar light irradiation. The CBZ degradation by NCDs/BiOBr/CeO2 could achieve 98% within 120 min illumination, which was much higher than BiOBr or BiOBr/CeO2. The enhanced degradation of CBZ was due to the fact that the introduction of NCDs significantly accelerated the migration and separation of the photoinduced charge carries at the tightly contacted interface. The effects of some key operating parameters were further investigated, including photocatalyst dosage, initial CBZ concentration, initial pH, coexisting inorganic anions (Cl−, NO3−, SO42−) and natural organic matters (NOM), light irradiation conditions and water matrix. Basing on the scavenging experiments and electron spin resonance (ESR), the photocatalytic mechanism was proposed for the charge separation. Furthermore, the transformation intermediates and possible degradation pathway of CBZ were investigated by liquid chromatography coupled with mass spectrometry (LC/MS) and three-dimensional excitation-emission matrix fluorescence spectra (3D EEMs). Finally, the composite showed excellent recyclability and stability. This study created new opportunities for the design of photocatalysts and degradation of recalcitrant organic contaminants in aquatic environment.

APOE Genotype Differentially Modulates Plasma Lipids in Healthy Older Individuals, with Relevance to Brain Health.

Apolipoprotein E (APOE) genotype is an established genetic risk factor for sporadic Alzheimer's disease (AD) but the extent to which APOE genotype influences the plasma lipidome is unknown, even though lipids are potential diagnostic or prognostic biomarkers for AD. We quantified plasma lipids using untargeted liquid chromatography coupled mass spectrometry in a total of 152 non-demented participants aged 65-100 years carrying at least one ɛ2 or ɛ4 allele (ɛ2/ɛ2 or ɛ2/ɛ3, n = 38: ɛ4/ɛ3 or ɛ4/ɛ4, n = 38), who were roughly matched to an ɛ3/ɛ3 control by age, sex, and lipid-lowering medication (n = 76). Low density lipoprotein cholesterol levels were genotype dependent (ɛ4/ɛ4> ɛ4/ɛ3> ɛ3/ɛ3> ɛ2/ɛ3> ɛ2/ɛ2). The greatest variation in lipids was related to the ɛ2 isoform, where various lysophosphatidylcholines and all phosphatidylethanolamine (PE) subclasses were elevated relative to ɛ3/ɛ3 and ɛ4 carriers. APOEɛ4 carriers had reduced phosphatidylinositol relative to ɛ3/ɛ3 and ɛ2 carriers. Logistic regression revealed that ɛ2 carriers were at least 4 times higher odds of being in the highest tertile of PE lipid level relative to ɛ3/ɛ3. The elevation in PE and other phospholipids in ɛ2 carriers may indicate the protective effect of ɛ2 is linked to these phospholipids. Additionally, high baseline PE in cognitively normal participants predicted protection against cognitive decline six years later. Our data suggest substantial modulation of plasma lipids by APOE genotype and therefore indicates possible lipid targets and pathomechanisms involved in AD risk.

Electrochemical degradation of aqueous alachlor and atrazine: products identification, lipophilicity, and ecotoxicity

This work studied the electrochemical degradation of alachlor and atrazine (alone and mixed with each other) using a filter-press cell, a dimensionally stable anode (DSA Ti/Ru0.3Ti0.7O2), initial pH 3.0, and temperature at 25 °C. The best operational conditions for alachlor (0.33 mmol L-1) degradation were obtained by a 32 factorial design, in which the factors/levels were: NaCl concentration (0.05, 0.1, and 0.15 mol L-1) and current density (10, 30, and 50 mA cm-2). Thus, 93.1% alachlor removal and 71.2% mineralization were achieved using 0.15 mol L-1 NaCl and 30 mA cm-2. In addition, the initial degradation products (DPs) of alachlor and atrazine were identified by liquid chromatography coupled to mass spectrometry (LC-MS). Acute and chronic ecotoxicities for three trophic levels (fishes, daphnids and green algae) and lipophilicity (log D, pH 7.4) of the DPs were also estimated using the ECOSAR 1.11 and ChemAxon Calculator software, respectively. The present study showed that the electrochemical degradation is an efficient method for removing the herbicides alachlor and atrazine from water and that the DPs formed have lower pollution potential than their original compounds.

Investigation of Aloe ferox leaf powder on anti-diabesity activity

Obesity and diabetes contribute largely to increased morbidity and mortality globally. However, the treatment of obesity is not well established, hence plant materials are being considered for the development of new anti-diabesity therapies. Aloe ferox has been used for different medicinal purposes, but the research on its anti-diabesity activity is limited. This study therefore aimed to investigate the anti-diabesity potential of Aloe ferox leaf powder. Sprague Dawley rats assigned into 2 groups consisting of 8 animals each were fed with either a high fat diet (HFD; control), or a high fat diet containing Aloe ferox leaf powder (HFD-AL; treatment) over a period of 12 weeks. The animals were weighed weekly and their food consumption was monitored three times a week. Blood samples were collected after 12 weeks and analysed for liver function, fructosamine level, cholesterol and triglycerides levels. Furthermore, the aqueous and 50% dichloromethane-methanol extracts of A. ferox leaf powder were also analysed for their α-amylase and α-glucosidase inhibition potential. Phytochemical analysis was done using Liquid Chromatography coupled with Mass Spectroscopy (LC-MS) to identify active compounds. Body weight gain was lower in animals on HFD-AL than HFD only. Fructosamine level was significantly lower in HFD-AL than HFD animals at p<0.05. HFD-AL animals also presented with lower cholesterol and triglycerides levels as well as less adipose tissue than animals in the control. Histology examination did not reveal any lesions due to toxic effects in any of the organs in both groups. Aloe ferox leaf extracts showed potential inhibition of α-glucosidase, with little effect on α-amylase inhibition in vitro assays. Aloe ferox leaf powder showed the presence of isoferulic acid-3-glucoronide, chrysophanol-8-O-glucoside, oleic acid and stearic acid. While Aloe ferox water extract showed the presence of isorhamnetin, cinnamic acid and caffeoylshikimic acid. Aloe ferox leaf powder showed potential to prevent weight gain and possible hypoglycaemic effect in animals fed with high fat diet. It also showed potential glucose modulation activity through inhibition of α-glucosidase enzyme activity.

Environmental exposures in young adults with declining kidney function in a population at risk of Mesoamerican nephropathy

ObjectivesThere is an epidemic of Mesoamerican nephropathy (MeN) in Central America, where sugarcane production is prominent. Numerous causes are proposed, but to date limited evidence supports any one hypothesis. A...

Phytochemical comparison of different tea (Camellia sinensis) cultivars and its association with sensory quality of finished tea

Selection of appropriate tea (Camellia sinensis) cultivars, represents an essential prerequisite for tea manufacture. A detailed phytochemical comparison of different cultivars, and its influence on flavor quality of finished tea, remain to be explored. Herein, different Chinese tea cultivars which are suited for only green or black tea processing (GT, BT cultivars), or compatible for both tea types processing (BT> cultivars), were pre-selected and carefully collected. A nontargeted metabolomics using liquid chromatography coupled to mass spectrometry (LC-MS) was performed, combined with comprehensive pathway mapping. Meanwhile, all cultivars were subjected to parallel processing for sensory evaluation of finished black tea. BT cultivars showed most prominent differences, with significantly higher levels of catechins, condensed products and phenolic acids including gallotannins, whereas lower contents of flavone C-glycosides, free sugars and amino acids were found. Particularly, unique glycosylation patterns of flavonol glycosides were identified for the first time. O-rutinosylation and O-glucosylation were predominant in BT cultivars, whereas O-glucosyl/galactosyl-rutinosylation were predominant in GT and BT> cultivars. The flavor attributes of differential compounds could be well associated with corresponding processing suitability of studied cultivars. This study provides an in-depth elucidation of metabolic features associated with processing suitability of different tea cultivars.

Sequencing Heparan Sulfate Using HILIC LC-NETD-MS/MS.

Heparan sulfate (HS) mediates a wide range of protein binding interactions key to normal and pathological physiology. Though liquid chromatography coupled with mass spectrometry (LC-MS) based disaccharide composition analysis is able to profile changes in HS composition, the heterogeneity of modifications and the labile sulfate group present major challenges for liquid chromatography tandem mass spectrometry (LC-MS/MS) sequencing of the HS oligosaccharides that represent protein binding determinants. Here, we report online LC-MS/MS sequencing of HS oligosaccharides using hydrophilic interaction liquid chromatography (HILIC) and negative electron transfer dissociation (NETD). A series of synthetic HS oligosaccharides varying in chain length (tetramers and hexamers), number of sulfate groups (3-7), sulfate patterns (sulfate positional isomers), and uronic acid epimerization (epimers) were separated and sequenced. The LC elution order of isomeric compounds was associated with their fine structure. The application of an online cation exchange device (ion suppressor) enhanced the precursor charge states, and the subsequent NETD produced abundant glycosidic fragments, allowing the characterization of both lowly sulfated and highly sulfated HS oligosaccharides. Furthermore, the diagnostic cross-ring ions differentiated the 6-O sulfation and 3-O sulfation, allowing unambiguous structural assignment. Collectively, this LC-NETD-MS/MS method is a powerful tool for sequencing of heterogeneous HS mixtures and is applicable for the differentiation of both isomers and epimers, for the characterization of saccharide mixtures with a varying extent of sulfation and even for the determination of both predominant and rare modification motifs. Thus, LC-NETD-MS/MS has great potential for further application to biological studies.

Metabolomic Profiling of the Host Response of Tomato (Solanum lycopersicum) Following Infection by Ralstonia solanacearum.

Tomato (Solanum lycopersicum) is an important dietary source of bioactive phytochemicals and active breeding programs constantly produce new cultivars possessing superior and desirable traits. The phytopathogenic Ralstonia solanacearum, the causal agent of bacterial wilt, is a highly destructive bacterial disease with a high economic impact on tomato production. This study followed an untargeted metabolomic approach involving four tomato cultivars and aimed at the identification of secondary metabolites involved in plant defense after infection with R. solanacearum. Liquid chromatography coupled to mass spectrometry (LC-MS) in combination with multivariate data analysis and chemometric modelling were utilized for the identification of discriminant secondary metabolites. The total of 81 statistically selected features were annotated belonging to the metabolite classes of amino acids, organic acids, fatty acids, various derivatives of cinnamic acid and benzoic acids, flavonoids and steroidal glycoalkaloids. The results indicate that the phenylpropanoid pathway, represented by flavonoids and hydroxycinnamic acids, is of prime importance in the tomato defense response. The hydroxycinnamic acids esters of quinic acid, hexoses and glucaric acids were identified as signatory biomarkers, as well as the hydroxycinnamic acid amides to polyamines and tyramine. Interestingly, the rapid and differential accumulation of putrescine, dopamine, and tyramine derivatives, along with the presence of a newly documented metabolite, feruloyl serotonin, were documented in the infected plants. Metabolite concentration variability in the different cultivar tissues point to cultivar-specific variation in the speed and manner of resource redistribution between the host tissues. These metabolic phenotypes provide insights into the differential metabolic signatures underlying the defense metabolism of the four cultivars, defining their defensive capabilities to R. solanacearum.

Liquid chromatography-coupled mass spectrometry analysis of glutathione conjugates of oxygenated polyunsaturated fatty acids

Glutathione (GSH) conjugates of oxygenated polyunsaturated fatty acids comprise a group of pro-inflammatory and pro-resolving lipid mediators formed in immunocompetent cells. While the pro-inflammatory conjugates such as the cysteinyl leukotrienes (cys-LTs), eoxins (EXs) and five-oxo-GSH conjugate (FOG7) derive from arachidonic acid (AA), the group of conjugates in tissue regeneration (CTRs) such as maresin CTRs (MCTRs), protectin CTRs (PCTRs) and resolvin CTRs (RCTRs) are biosynthesized from docosahexaenoic acid (DHA). Here, we present a gradient UPLC-MS/MS method for the analysis of pro-inflammatory and pro-resolving GSH conjugates using positive electrospray ionization (ESI(+)) and collision-induced fragmentation for unambiguous identification and structural information, and a negative ionization (ESI(-)) mode for quantification of the GSH conjugates. The method was employed to detect GSH conjugates in human platelets and macrophages. MCTRs were detected in platelets upon addition of exogenous docosahexaenoic acid (DHA) and the biosynthesis was independent on leukotriene C4 (LTC4) synthase activity. Pathogenic bacteria stimulated the formation of EXs and PCTRs in M2 macrophages, whereas Ca2+-ionophore activated the biosynthesis of LTC4 in M1 and M2 macrophage phenotypes. Together, our methodology covers the qualitative and quantitative analysis of GSH conjugates and gives an analytical basis for the detection and structural elucidation of cysteinyl-containing lipid mediators.