Liquid Chromatography-mass Spectrometry Approach Research Articles

Production of N-acyl homoserine lactones by the sponge-associated marine actinobacteria Salinispora arenicola and Salinispora pacifica.

The structures of acyl homoserine lactone (AHL) compounds and their quantification were accomplished using an integrated liquid chromatography-mass spectrometry approach. The precursor and product ions, along with retention times of peaks, were searched against an in-house database of AHLs and structures confirmed by accurate mass and by comparison with authentic AHL standards. The two compounds, N-(3-oxodecanoyl)-L-homoserine lactone and N-(3-oxododecanoyl)-L-homoserine lactone, were characterised and quantified in Salinispora sp. cultures.

Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS.

Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using N-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.

Development of a Rapid Microbore Metabolic Profiling Ultraperformance Liquid Chromatography-Mass Spectrometry Approach for High-Throughput Phenotyping Studies.

A rapid gradient microbore ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method has been developed to provide a high-throughput analytical platform for the metabolic phenotyping of urine from large sample cohorts. The rapid microbore metabolic profiling (RAMMP) approach was based on scaling a conventional reversed-phase UPLC-MS method for urinary profiling from 2.1 mm × 100 mm columns to 1 mm × 50 mm columns, increasing the linear velocity of the solvent, and decreasing the gradient time to provide an analysis time of 2.5 min/sample. Comparison showed that conventional UPLC-MS and rapid gradient approaches provided peak capacities of 150 and 50, respectively, with the conventional method detecting approximately 19 000 features compared to the ∼6 000 found using the rapid gradient method. Similar levels of repeatability were seen for both methods. Despite the reduced peak capacity and the reduction in ions detected, the RAMMP method was able to achieve similar levels of group discrimination as conventional UPLC-MS when applied to rat urine samples obtained from investigative studies on the effects of acute 2-bromophenol and chronic acetaminophen administration. When compared to a direct infusion MS method of similar analysis time the RAMMP method provided superior selectivity. The RAMMP approach provides a robust and sensitive method that is well suited to high-throughput metabonomic analysis of complex mixtures such as urine combined with a 5-fold reduction in analysis time compared with the conventional UPLC-MS method.

Anti-acetylcholinesterase potential and metabolome classification of 4 Ocimum species as determined via UPLC/qTOF/MS and chemometric tools

Ocimum (sweet basil) is a plant of considerable commercial importance in traditional medicine worldwide as well as for the flavor and food industry. The goal of this study was to examine Ocimum extracts anti-acetylcholinesterase activity and to correlate the activity with their secondary metabolites profiles via a metabolome based ultraperformance liquid chromatography-mass spectrometry (UPLC–MS) approach coupled to chemometrics. The metabolomic differences in phenolics from leaves derived from 4 Ocimum species: Ocimum basilicum, Ocimum africanum, Ocimum americanum and Ocimum minimum were assessed. Under optimized conditions, 81 metabolites were identified including 21 hydroxy cinnamic acids, 4 benzoic acid conjugates, 14C/O flavonoid conjugates, 2 alcohols, 5 acyl sugars, 4 triterpenes and 12 fatty acids. Several salviolanic acid derivatives including salviolanic acid A, B, C & I found in Salvia, were found in Ocimum herein for the first time. Unsupervised principal component analysis (PCA) and supervised orthogonal projection to latent structures-discriminant analysis (OPLS-DA) were further used for comparing and classification of samples. A clear separation among the four investigated Ocimum species was revealed, with O. africanum samples found most enriched in hydroxy cinnamates conjugates (HC) and flavonoids. To the best of our knowledge, this is the first report for compositional differences among Ocimum leaves via a metabolomic approach revealing that among examined species O. africanum leaves present a better source of Ocimum bioactive metabolites. The anticholinesrase activity of examined species was further assessed with a potent IC50 values for O. americanum, O. africanum, O. basilicum ranging from 2.5 to 6.6mg/ml, whereas O. minimum was least active with IC50 of 31.4mg/ml. Furthermore, major HC i.e., caftaric, chlorogenic and rosmarinic acids identified in extracts via UPLC–MS analysis exhibited IC50 values of 24, 0.5 and 7.9mg/ml respectively, suggesting that HCs are likely to mediate for the anticholinesterase effect in Ocimum extracts.

Untargeted Metabolomics To Ascertain Antibiotic Modes of Action.

Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5′-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5′-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.

The effect of elevated CO2 and virus infection on the primary metabolism of wheat.

Atmospheric CO2 concentrations are predicted to double by the end of this century. Although the effects of CO2 fertilisation in crop systems have been well studied, little is known about the specific interactions among plants, pests and pathogens under a changing climate. This growth chamber study focuses on the interactions among Barley yellow dwarf virus (BYDV), its aphid vector (Rhopalosiphum padi) and wheat (Triticum aestivum L. cv. Yitpi) under ambient (aCO2; 400µmolmol-1) or elevated (eCO2; 650µmolmol-1) CO2 concentrations. eCO2 increased the tiller number and biomass of uninoculated plants and advanced the yellowing symptoms of infected plants. Total foliar C content (percentage of the total DW) increased with eCO2 and with sham inoculation (exposed to early herbivory), whereas total N content decreased with eCO2. Liquid chromatography-mass spectrometry approaches were used to quantify the products of primary plant metabolism. eCO2 significantly increased sugars (fructose, mannitol and trehalose), irrespective of disease status, whereas virus infection significantly increased the amino acids essential to aphid diet (histidine, lysine, phenylalanine and tryptophan) irrespective of CO2 concentration. Citric acid was reduced by both eCO2 and virus infection. Both the potential positive and negative biochemical impacts on wheat, aphid and BYDV interactions are discussed.

Metformin Improves Diabetic Bone Health by Re-Balancing Catabolism and Nitrogen Disposal.

ObjectiveMetformin, a leading drug used to treat diabetic patients, is reported to benefit bone homeostasis under hyperglycemia in animal models. However, both the molecular targets and the biological pathways affected by metformin in bone are not well identified or characterized. The objective of this study is to investigate the bioengergeric pathways affected by metformin in bone marrow cells of mice.Materials and MethodsMetabolite levels were examined in bone marrow samples extracted from metformin or PBS -treated healthy (Wild type) and hyperglycemic (diabetic) mice using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. We applied an untargeted high performance LC-MS approach which combined multimode chromatography (ion exchange, reversed phase and hydrophilic interaction (HILIC)) and Orbitrap-based ultra-high accuracy mass spectrometry to achieve a wide coverage. A multivariate clustering was applied to reveal the global trends and major metabolite players.ResultsA total of 346 unique metabolites were identified, and they are grouped into distinctive clusters that reflected general and diabetes-specific responses to metformin. As evidenced by changes in the TCA and urea cycles, increased catabolism and nitrogen waste that are commonly associated with diabetes were rebalanced upon treatment with metformin. In particular, we found glutamate and succinate whose levels were drastically elevated in diabetic animals were brought back to normal levels by metformin. These two metabolites were further validated as the major targets of metformin in bone marrow stromal cells.ConclusionOverall using limited sample size, our study revealed the metabolic pathways modulated by metformin in bones which have broad implication in our understanding of bone remodeling under hyperglycemia and in finding therapeutic interventions in mammals.

Development and Validation of a High-Throughput Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Approach for Screening of Oxylipins and Their Precursors

Lipid mediators, highly bioactive compounds synthesized from polyunsaturated fatty acids (PUFAs), have a fundamental role in the initiation and signaling of the inflammatory response. Although extensively studied in isolation, only a limited number of analytical methods offer a comprehensive coverage of the oxylipin synthetic cascade applicable to a wide range of human biofluids. We report the development of an ultrahigh-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UHPLC-MS) assay to quantify oxylipins and their PUFA precursors in 100 μL of human serum, plasma, urine, and cell culture supernatant. A single 15 min UHPLC run enables the quantification of 43 oxylipins and 5 PUFAs, covering pro and anti-inflammatory lipid mediators synthesized across the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) pathways. The method was validated in multiple biofluid matrixes (serum, plasma, urine, and cell supernatant) and suppliers, ensuring its suitability for large scale metabonomic studies. The approach is accurate, precise, and reproducible (RSD < 15%) over multiple days and concentrations. Very high sensitivity is achieved with limits of quantification inferior to picograms for the majority of analytes (0.05-125 pg) and linear range spanning up to 5 orders of magnitude. This enabled the quantification of the great majority of these analytes at their low endogenous level in human biofluids. We successfully applied the procedure to individuals undergoing a fasting intervention; oxylipin profiles highlighted significantly altered PUFA and inflammatory profiles in accordance with previously published studies as well as offered new insight on the modulation of the biosynthetic cascade responsible for the regulation of oxylipins.

The Siderophore Metabolome of Azotobacter vinelandii.

In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.

An overview of methods using (13)C for improved compound identification in metabolomics and natural products.

Compound identification is a major bottleneck in metabolomics studies. In nuclear magnetic resonance (NMR) investigations, resonance overlap often hinders unambiguous database matching or de novo compound identification. In liquid chromatography-mass spectrometry (LC-MS), discriminating between biological signals and background artifacts and reliable determination of molecular formulae are not always straightforward. We have designed and implemented several NMR and LC-MS approaches that utilize 13C, either enriched or at natural abundance, in metabolomics applications. For LC-MS applications, we describe a technique called isotopic ratio outlier analysis (IROA), which utilizes samples that are isotopically labeled with 5% (test) and 95% (control) 13C. This labeling strategy leads to characteristic isotopic patterns that allow the differentiation of biological signals from artifacts and yield the exact number of carbons, significantly reducing possible molecular formulae. The relative abundance between the test and control samples for every IROA feature can be determined simply by integrating the peaks that arise from the 5 and 95% channels. For NMR applications, we describe two 13C-based approaches. For samples at natural abundance, we have developed a workflow to obtain 13C–13C and 13C–1H statistical correlations using 1D 13C and 1H NMR spectra. For samples that can be isotopically labeled, we describe another NMR approach to obtain direct 13C–13C spectroscopic correlations. These methods both provide extensive information about the carbon framework of compounds in the mixture for either database matching or de novo compound identification. We also discuss strategies in which 13C NMR can be used to identify unknown compounds from IROA experiments. By combining technologies with the same samples, we can identify important biomarkers and corresponding metabolites of interest.

Serum Dyslipidemia Is Induced by Internal Exposure to Strontium-90 in Mice, Lipidomic Profiling Using a Data-Independent Liquid Chromatography-Mass Spectrometry Approach.

Despite considerable research into the environmental risks and biological effects of exposure to external beam γ rays, incorporation of radionuclides has largely been understudied. This dosimetry and exposure risk assessment is challenging for first responders in the field during a nuclear or radiological event. Therefore, we have developed a workflow for assessing injury responses in easily obtainable biofluids, such as urine and serum, as the result of exposure to internal emitters cesium-137 ((137)Cs) and strontium-90 ((90)Sr) in mice. Here we report on the results of the untargeted lipidomic profiling of serum from mice exposed to (90)Sr. We also compared these results to those from previously published (137)Cs exposure to determine any differences in cellular responses based on exposure type. The results of this study conclude that there is a gross increase in the serum abundance of triacylglycerides and cholesterol esters, while phostaphatidylcholines and lysophosphatidylcholines displayed decreases in their serum levels postexposure at study days 4, 7, 9, 25, and 30, with corresponding average cumulative skeleton doses ranging from 1.2 ± 0.1 to 5.2 ± 0.73 Gy. The results show significant perturbations in serum lipidome as early as 2 days postexposure persisting until the end of the study (day 30).

Electrospray ionization linear trap quadrupole Orbitrap in analysis of old tempera paintings: application to nineteenth-century Orthodox icons.

Proteomic approach in combination with mass spectrometry demonstrates a great potential for identification of proteinaceous materials in works of art. In this study we used a linear trap quadrupole Orbitrap (LTQ-Orbitrap), a state-of-the-art mass spectrometer for parts per million accuracy analyses of peptides behind tryptic hydrolysis. After the efficiency of the proteomic method was confirmed for reference and model samples, micro-samples from historical paintings were for the first time analysed using this technique. Superior performances of the liquid chromatography-mass spectrometry approach using a LTQ-Orbitrap mass spectrometer allowed identification of egg yolk peptides in two samples from nineteenth-century Orthodox icons, indicating egg tempera as the painting technique. Accurate precursor ion masses, in the range of ±2ppm, and retention times of tryptic peptides strengthen protein identification. Additionally, in all historical samples the presence of animal glues suggested that the ground layer was likely bound using bovine collagen. Comparing to results acquired using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry in our previous study, here we achieved higher ion scores and protein scores, better sequence coverage and more identified proteins. In fact, a combination of the two mass spectrometric techniques provided overlapping and complementary data, related to the detection of peptides with different physicochemical properties.

Whole Grain Consumption Increases Gastrointestinal Content of Sulfate-Conjugated Oxylipins in Pigs - A Multicompartmental Metabolomics Study.

The effects of increased intake of dietary fiber as either whole grain rye or as resistant starch compared to a typical low dietary fiber Western-style diet on the metabolomics responses were studied in gastrointestinal content and tissue, peripheral plasma, and urine using a multicompartmental nontargeted liquid chromatography-mass spectrometry (LC-MS) approach in pigs. Both unsupervised and supervised multivariate analyses were used to study differences between the intervention groups, revealing significant effects of the dietary intervention on gastrointestinal contents and urine metabolites. Consumption of a diet composed of whole grain rye flakes and enzyme treated wheat bran increased gastrointestinal content of novel sulfate- and acetic acid-conjugated oxylipins, and urinary secretion of phenolic compounds. Furthermore, potential gastrointestinal and urinary biomarkers for consumption of resistant starch, whole grain rye, and a low dietary fiber intake were detected using multiblock analysis. This study provides insight into microbial fermentation products in the gastrointestinal tract and suggests a potential role in sulfate conjugation of metabolites on the bioavailability of ingested nutrients.

Determination of oxidized phosphatidylcholines by hydrophilic interaction liquid chromatography coupled to Fourier transform mass spectrometry.

A novel liquid chromatography-mass spectrometry (LC-MS) approach for analysis of oxidized phosphatidylcholines by an Orbitrap Fourier Transform mass spectrometer in positive electrospray ionization (ESI) coupled to hydrophilic interaction liquid chromatography (HILIC) was developed. This method depends on three selectivity criteria for separation and identification: retention time, exact mass at a resolution of 100,000 and collision induced dissociation (CID) fragment spectra in a linear ion trap. The process of chromatography development showed the best separation properties with a silica-based Kinetex column. This type of chromatography was able to separate all major lipid classes expected in mammalian samples, yielding increased sensitivity of oxidized phosphatidylcholines over reversed phase chromatography. Identification of molecular species was achieved by exact mass on intact molecular ions and CID tandem mass spectra containing characteristic fragments. Due to a lack of commercially available standards, method development was performed with copper induced oxidation products of palmitoyl-arachidonoyl-phosphatidylcholine, which resulted in a plethora of lipid species oxidized at the arachidonoyl moiety. Validation of the method was done with copper oxidized human low-density lipoprotein (LDL) prepared by ultracentrifugation. In these LDL samples we could identify 46 oxidized molecular phosphatidylcholine species out of 99 possible candidates.

Schistosome and liver fluke derived catechol-estrogens and helminth associated cancers.

Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.

Identification of new flavonols in hybrid grapes by combined liquid chromatography–mass spectrometry approaches

Grape flavonols are involved in the phenomenon of copigmentation in red wines. These compounds are characterised by nutraceutical properties, have antioxidant activity and are studied for chemotaxonomy of grapes. In general, hybrid grapes are characterised by presence of polyphenols often qualitatively and quantitatively different from Vitis vinifera varieties. In this work, flavonols of 34 hybrid grape varieties (22 red and 12 white) produced by crossing of V. vinifera, Vitis riparia, Vitis labrusca, Vitis lincecumii and Vitis rupestris species, were studied. Compounds were characterised by combining different liquid chromatography/mass spectrometry (LC/MS) methods: precursor-ion and neutral-loss multiple-reaction-monitoring (MRM), and high-resolution mass spectrometry. Twenty-four glycoside flavonols were identified, including 4 quercetin, 5 myricetin, 4 kaempferol, 3 isorhamnetin, 2 laricitrin, 3 syringetin and 3 dihydroflavonol derivatives; myricetin hexoside-glucuronide, myricetin O-di-hexoside, syringetin O-di-hexoside, isorhamnetin rutinoside and kaempferol rutinoside were found in grape for the first time. Statistical analysis (PCA and cluster analysis) divided the samples in four groups on the basis of their flavonol profiles.

Identification and Pharmacokinetics of Novel Alkylresorcinol Metabolites in Human Urine, New Candidate Biomarkers for Whole-Grain Wheat and Rye Intake1–3

Biomarkers of dietary intake are prominent tools in nutritional research. The alkylresorcinol metabolites 3,5-dihydroxybenzoic acid (3,5-DHBA) and 3-(3,5-dihydroxyphenyl)propanoic acid (3,5-DHPPA) have been proposed as exposure biomarkers of whole-grain (WG) wheat and rye intake. However, the profile of alkylresorcinol metabolites is not fully understood. The aim of this study was to investigate the metabolism of alkylresorcinols in mice and in humans, while further determining urinary pharmacokinetics of the novel alkylresorcinol metabolites to explore their potential as biomarkers of WG wheat intake. Utilization of the liquid chromatography–mass spectrometry approach resulted in 10 alkylresorcinol metabolites identified in mice and in humans, including 3 phenolic acids and 7 of their phase II conjugates. Among them, 2 novel metabolites were discovered: 5-(3,5-dihydroxyphenyl)pentanoic acid (3,5-DHPPTA) and 2-(3,5-dihydroxybenzamido)acetic acid (3,5-DHBA glycine). The structures of these 2 metabolites were confirmed by comparing with authentic standards synthesized in-house. In the pharmacokinetic study, a group of 12 volunteers consumed a polyphenolic-restricted diet for 4 d before ingesting WG wheat bread containing 61 mg of alkylresorcinols. Urine samples were collected for 32 h, and alkylresorcinol metabolites were quantified with HPLC–coulometric electrode array detection. The mean urinary excretion rates and mean apparent half-life of 3,5-DHPPTA, 3,5-DHBA glycine, 3,5-DHBA, and 3,5-DHPPA at each time point were determined. Our results suggest that 3,5-DHPPTA and 3,5-DHBA glycine may be used in combination with 3,5-DHBA and 3,5-DHPPA as potential biomarkers to increase the accuracy of recording WG wheat and rye intake in epidemiologic studies. Further validation of 3,5-DHPPTA and 3,5-DHBA glycine as potential biomarkers is warranted.

A serum metabolomics study of gastric cancer based on pseudotargeted liquid chromatography-mass spectrometry approach

Gastric cancer is one of the most common malignant and the second most frequent cause of cancer-related mortality in the world. Noninvasive screen methods including tumor markers are intensively needed in the clinic. To discover metabolic markers for gastric cancers, the sera metabolic profiles of 20 gastric cancer (GC) patients and 40 healthy controls were analyzed by liquid chromatography coupled with mass spectrometry (LC-MS) using a pseudotargeted approach. The results of principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) indicated that obvious classification could be observed between GC and the healthy controls. According to the results of non-parametric statistical test, 57 ions were identified among all the differential metabolic features. Of all the identified differential metabolites, dihydrocholesterol provide good diagnostic performance of GC, which was also validated by another cohort including the 20 gastric cancer patients and the 39 healthy controls. With this robust pseudotargeted approach, our study provides both potential tumor markers and the basis for the metabolic phenotype of gastric cancers.

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

Extracytoplasmic Proteases Determining the Cleavage and Release of Secreted Proteins, Lipoproteins, and Membrane Proteins in Bacillus subtilis

Gram-positive bacteria are known to export many proteins to the cell wall and growth medium, and accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a gel electrophoresis-based liquid chromatography-mass spectrometry approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental "protein export stresses" upon overproduction of membrane or secreted proteins. Our results show that many secreted proteins, lipoproteins, and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under nonstress conditions.