Metabolic profiling of gender: Headspace-SPME/GC–MS and <Superscript>1</Superscript>H NMR analysis of urine

This study aims to investigate the metabolic difference between male and female healthy adults using a combination of GC–MS and NMR metabolomics techniques. While metabolomics has shown wide applications in characterizing the status and progression of many diseases, physiological factors such as gender often contribute high levels of variability that can hinder the detection of biomarkers of interest, such as in disease detection. We carried out a detailed exploration of gender related metabolic profiling of human urine using a Headspace-SPME/GC–MS approach and detected over two hundred peaks. Fifty-nine metabolites were identified using the NIST library. ¹H NMR spectroscopy was also utilized, and resulted in the identification of eighteen metabolites. We find that both GC–MS and NMR are able to capture human gender metabolic differences, and their combination allows a significantly better understanding of this difference. Subtle differences between genders are found to be related to the metabolism of fats, amino acids, and TCA cycle intermediates.     

Evaluation of oxysterol levels of patients with silicosis by LC–MS/MS method

Molecular and Cellular Biochemistry - Aberrant structural formations of Cu/Zn superoxide dismutase enzyme (SOD1) are the probable mechanism by which circumscribed mutations in the SOD1 gene cause...     

Diagnosis of <Emphasis Type="Italic">Clostridium difficile</Emphasis> infection using an UPLC–MS based metabolomics method

Introduction

The fecal metabolome of Clostridium difficile (CD) infection is far from being understood, particularly its non-volatile organic compounds. The drawbacks of current tests used to diagnose CD infection hinder their application.

Objective

The aims of this study were to find new characteristic fecal metabolites of CD infection and develop a metabolomics model for the diagnosis of CD infection.

Methods

Ultra-performance liquid chromatography-mass spectrometry (UPLC–MS) was used to characterize the fecal metabolome of CD positive and negative diarrhea and healthy control stool samples.

Results

Diarrhea and healthy control samples showed distinct clusters in the principal components analysis score plot, and CD positive group and CD negative group demonstrated clearer separation in a partial least squares discriminate analysis model. The relative abundance of sphingosine, chenodeoxycholic acid, phenylalanine, lysophosphatidylcholine (C16:0), and propylene glycol stearate was higher, and the relative abundance of fatty amide, glycochenodeoxycholic acid, tyrosine, linoleyl carnitine, and sphingomyelin was lower in CD positive diarrhea groups, than in the CD negative group. A linear discriminant analysis model based on capsiamide, dihydrosphingosine, and glycochenodeoxycholic acid was further constructed to identify CD infection in diarrhea. The leave-one-out cross-validation accuracy and area under receiver operating characteristic curve for the training set/external validation set were 90.00/78.57%, and 0.900/0.7917 respectively.

Conclusions

Compared with other hospital-onset diarrhea, CD diarrhea has distinct fecal metabolome characteristics. Our UPLC–MS metabolomics model might be useful tool for diagnosing CD diarrhea.

     

Metabolomic study of the bone trabecula of osteonecrosis femoral head patients based on UPLC–MS/MS

The severity and/or progression of osteonecrosis of the femoral head (ONFH) are commonly assessed by radiography, nuclear magnetic resonance image which aren’t invariably correlated to severity of disease and may be disturbed by other factors. Consequently, exploring the novel biochemical signatures of ONFH may be beneficial for diagnosing and understanding this disease. In this work, a bone trabecula metabolomics was undertaken to determine the expression pattern of low molecular mass metabolites in patients of femoral head necrosis based on the ultra-performance liquid chromatography/time-of-flight tandem mass spectrometry (UPLC/TOF MS/MS). Histological study showed that necrotic bone was characterized by necrosis, fibrosis and lacuna, but adjacent “normal” bone was pathologically normal. Principal component analysis in combination with orthogonal partial least-squares discrimination analysis was used to find out changed metabolites. MS/MS was used to speculate the corresponding molecule. Both osteonecrotic bone trabecula (ONBT) and adjacent “normal” bone trabecula (ANBT) showed higher levels of amino acids, such as proline, arginine, glutamine, dipeptides and lower levels of antioxidants. Most disrupted lipids, such as fatty acid esters, glycerophospholipids, sphingolipids, were found in osteonecrotic zone. The area under the receiver operating characteristic curve of combinational biomarkers (d-arginine, l-proline, l-carnitine, inosine) in ONBT and ANBT was 0.996 and 0.950, respectively. Our findings might provide a significant insight to understand the metabolic mechanism and diagnosis biomarkers of ONFH in the future.     

Effect of various culture conditions on shoot multiplication and GC–MS analysis of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> accessions for plumbagin production

Plumbago zeylanica, a pharmaceutically important medicinal plant, contains a wide range of phytocompounds. Culture parameters like carbon source, nitrogen source, and culture media are essential for the development and growth of explants. In this investigation, the influence of various carbon sources (sucrose, glucose, and fructose at 3% concentration), nitrogen source (ammonium nitrate, sodium nitrate, and potassium nitrate) and plant tissue culture media (MS medium, Gamborg’s B5 medium, White medium and Nitsch medium) on shoot multiplication of five different accessions was studied. Optimum growth of all five accessions was observed in MS media containing 3% sucrose and ammonium nitrate as a source of carbon and nitrogen. Out of five accessions, IC-524441 showed the highest shoot multiplication. Further, methanolic extracts of all accessions (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source) were prepared and comparison of extracts in DPPH assay indicated that accession number IC-524441 was the most effective free radical scavenging agent. Total phenolic, flavonoid and tannin content ranges were from 20 to 70 µg/ml, 40 to 100 µg/ml and 55 to 120 µg/ml, respectively, and the highest amount was found in accession number IC-524441. Sucrose and ammonium nitrate content may be responsible for increased antioxidant activity, flavonoids content, phenolic content, and tannin content in accession number IC-524441. GC–MS of ethyl acetate extract of all five accessions of P. zeylanica was conducted (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source). GC–MS analysis of the aerial part showed the presence of various phytocompounds, which include 1,4-naphthalenedione, 3-eicosene, 5-eicosene, phthalic acid, o-anisic acid, thioctic acid, 1-octadecene, 5-t-butyl-cycloheptene, 2-benzoyl-1,2-dihydro-1-isoquinolinecarbonitrile, octadecanal, silane, 3-methoxy-2-methyl-2-(1-phenyl-ethylamino)-propionic acid, and 1-nonadecene. Accession number IC-524441 contains the highest amount of plumbagin, i.e. 14.19?±?0.5 µg/ml as compared to the others.     

GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-¹⁵N₂]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.     

Proteome profile of bovine ruminal epithelial tissue based on GeLC–MS/MS

The proteome of rumen epithelial tissue was analysed by SDS-PAGE coupled with LC–MS/MS. 813 non-redundant proteins were identified of which 7.4 % featured membrane-spanning domains and 15.4 % harboured a signal peptide. According to the gene ontology annotation, the most abundant proteins exhibited binding activities related to their molecular functions, were proteins of cellular components or belonged to various metabolic processes. A predominant group of canonical pathways in the rumen epithelial tissue was identified using the IPA software. The GeLC–MS/MS approach was used to characterise the entire protein expression repertoire in rumen tissue, providing a more detailed understanding of the important biological processes in the rumen.     

NMR-based metabolomic analysis of wild,greenhouse, and in vitro regenerated shoots of <Emphasis Type="Italic">Cymbopogon schoenanthus</Emphasis> subsp. <Emphasis Type="Italic">proximus</Emphasis> with GC–MS assessment of proximadiol

Cymbopogon schoenanthus subsp. proximus is a wild plant distributed in subtropical and east Africa extending from the north to the southern parts of Egypt. Widely used in folk medicine, it is the source of the diuretic sesquiterpene proximadiol. Nuclear magnetic resonance metabolomic analysis of polar extracts of shoots from wild, greenhouse, somatic embryos, and direct and indirect organogenic in vitro cultures was carried out. Metabolic profiling yielded 39 compounds, of which common metabolites were 15 (38.4%). Unique metabolites were trehalose (2.5%) in the wild plants, 2-hydroxylisobutyrate, galactarate and tyrosine (7.6%) in indirect organogenic shoots. Tartrate was found only in direct regenerated shoots (2.5%). Metabolites identified in greenhouse and embryogenic shoots showed no unique compounds. Multivariate analysis revealed significant differences between all tested shoots. 4-aminobutyrate, alanine, glutamine, glucose, fructose, and sucrose were the most significantly different metabolites. Proximadiol was identified and quantitatively measured from the non-polar extract of different types of shoots using gas chromatography and mass spectrometry (GC–MS). Concentrations ranged from 3.6 ± 0.03 to 198.6 ± 7.2 µg/100 mg dry weight in regenerated shoots from somatic embryogenesis and in wild plant shoots, respectively. Direct organogenesis yielded the highest in vitro concentration (20.3 ± 0.5 µg/100 mg dry weight). This study reported the metabolic profiling of C. schoenanthus polar extract and identified primary metabolites that are unique to the wild type and shoots regenerated from different in vitro cultures. Proximadiol was quantified and the in vitro culture system yielding the highest concentration relative to the wild plant was identified.     

Metabolic profiling of <Emphasis Type="Italic">Daphnia magna</Emphasis> exposed to environmental stressors by GC–MS and chemometric tools

Introduction

Climate change is a major concern for the scientific community, demanding novel information about the effects of environmental stressors on living organisms. Metabolic profiling is required for achieving the most extensive possible range of compounds and their concentration changes on stressed conditions.

Objectives

Individuals of the crustacean species Daphnia magna were exposed to three different abiotic factors linked to global climate change: high salinity, high temperature levels and hypoxia. Advanced chemometric tools were used to characterize the metabolites affected by the exposure.

Method

An exploratory analysis of gas chromatography-mass spectrometry (GC–MS) data was performed to discriminate between control and exposed daphnid samples. Due to the complexity of these GC–MS data sets, a comprehensive untargeted analysis of the full scan data was performed using multivariate curve resolution-alternating least squares (MCR-ALS) method. This approach enabled to resolve most of the metabolite signals from interference peaks caused by derivatization reactions. Metabolites with significant changes in their peak areas were tentatively identified and the involved metabolic pathways explored.

Results

D. magna metabolic biomarkers are proposed for the considered physical factors. Metabolites related with energy metabolic pathways including some amino acids, carbohydrates, organic acids and nucleosides were identified as potential biomarkers of the investigated treatments.

Conclusions

The proposed untargeted GC–MS metabolomics strategy and multivariate data analysis tools were useful to investigate D. magna metabolome under environmental stressed conditions.

     

LC–MS/MS-Based Metabolome Analysis of Biochemical Pathways Altered by Food Limitation in Larvae of Ivory Shell, <Emphasis Type="Italic">Babylonia areolata</Emphasis>

Ivory shell, Babylonia areolata, is one of the commercially important mariculture species in China and South East Asia. Survival varies in the artificial hatching and larval rearing of B. areolata. Food deprivation may be involved in rearing mortality, and so, a better understanding of how larvae respond and adjust to starvation is needed. In this study, the metabolite profiles of newly hatched larvae with yolk (I), larvae with yolk exhaustion (II), larvae suffering 24 h starvation after yolk exhaustion (III), and larvae fed with exogenous nutrients after yolk exhaustion (IV) were analyzed by LC–MS/MS. Principal component and cluster analyses revealed differential abundance of metabolite profiles across groups. When compared to metabolite levels of the I group, significantly up-regulated metabolites included polyunsaturated fatty acids, phospholipids, nucleotide, amino acids, and their derivatives were found in the II group, indicating that organisms relied predominantly on glycerophospolipid metabolism and protein-based catabolism for energy production during this stage. During starvation after yolk exhaustion, the levels of all energy related metabolites were significantly reduced, but an increase in products of purine and pyrimidine metabolism indicated an insufficient energy supply and an increase in cellular disintegration. Larvae fed exogenous nutrients can have significantly improved metabolism compared to starved larvae. These findings suggest that metabolomics, using LC–MS/MS, can be used to assess the physiological status and food-affected metabolic changes affecting B. areolata larvae.     

Identification of oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas 42A2

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC–MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (μg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64.     

Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC–MS/MS analysis

In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC–MS/MS (nanoLC–MS^E) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood.     

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂ and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.     

Efficient analysis and extraction of MS/MS result data from Mascot™ result files

Background  

Mascot™ is a commonly used protein identification program for MS as well as for tandem MS data. When analyzing huge shotgun proteomics datasets with Mascot™'s native tools, limits of computing resources are easily reached. Up to now no application has been available as open source that is capable of converting the full content of Mascot™ result files from the original MIME format into a database-compatible tabular format, allowing direct import into database management systems and efficient handling of huge datasets analyzed by Mascot™.     

TD/GC–MS analysis of volatile markers emitted from mono- and co-cultures of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in artificial sputum

Introduction

Infections such as ventilator-associated pneumonia (VAP) can be caused by one or more pathogens. Current methods for identifying these pathogenic microbes often require invasive sampling, and can be time consuming, due to the requirement for prolonged cultural enrichment along with selective and differential plating steps. This results in delays in diagnosis which in such critically ill patients can have potentially life-threatening consequences. Therefore, a non-invasive and timely diagnostic method is required. Detection of microbial volatile organic compounds (VOCs) in exhaled breath is proposed as an alternative method for identifying these pathogens and may distinguish between mono- and poly-microbial infections.

Objectives

To investigate volatile metabolites that discriminate between bacterial mono- and co-cultures.

Methods

VAP-associated pathogens Enterobacter cloacae and Pseudomonas aeruginosa were cultured individually and together in artificial sputum medium for 24 h and their headspace was analysed for potential discriminatory VOCs by thermal desorption gas chromatography–mass spectrometry.

Results

Of the 70 VOCs putatively identified, 23 were found to significantly increase during bacterial culture (i.e. likely to be released during metabolism) and 13 decreased (i.e. likely consumed during metabolism). The other VOCs showed no transformation (similar concentrations observed as in the medium). Bacteria-specific VOCs including 2-methyl-1-propanol, 2-phenylethanol, and 3-methyl-1-butanol were observed in the headspace of axenic cultures of E. cloacae, and methyl 2-ethylhexanoate in the headspace of P. aeruginosa cultures which is novel to this investigation. Previously reported VOCs 1-undecene and pyrrole were also detected. The metabolites 2-methylbutyl acetate and methyl 2-methylbutyrate, which are reported to exhibit antimicrobial activity, were elevated in co-culture only.

Conclusion

The observed VOCs were able to differentiate axenic and co-cultures. Validation of these markers in exhaled breath specimens could prove useful for timely pathogen identification and infection type diagnosis.

     

Application of liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the analysis of stable isotope enrichments of phenylalanine and tyrosine

Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the measurement of very low tracer–tracee ratios (TTR) of the amino acids l-phenylalanine and l-tyrosine in human plasma. TTR calibration curves of the tracers l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine were linear (r² > 0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 μM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean ± SD) of 3.33 ± 0.19% for l-[ring-²H₅]-phenylalanine, 2.40 ± 0.43% for l-[ring-²H₂]-tyrosine and 0.29 ± 0.07% for l-[ring-²H₄]-tyrosine, respectively. The LC–MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.     

Structure of the δ-Chain of Chimpanzee Haemoglobin A<Subscript>2</Subscript>

STUDIES of adult¹ and foetal² haemoglobin from the chimpanzee (Pan troglodytes) have shown that the amino-acid compositions of tryptic and chymotryptic peptides of the α, β and γ-chains are indistinguishable from those of man. The primary structures of chimpanzee α, β and γ-chains are therefore almost certainly identical to the homologous human chains. The two types of γ-chains found in man³, ^Gγ and ^Aγ, with glycine and alanine in position γ136, respectively, are likewise present in the chimpanzee².     

iTRAQ-Coupled 2D LC–MS/MS Analysis of Secreted Proteome of HBV-Replicating HepG2 Cells: Potential in Biomarkers for Prognosis of HCC

Hepatitis B virus (HBV) infection remains a major health concern with more than 350 million carriers in the world. It is associated with acute and chronic liver diseases including hepatocellular carcinoma (HCC). The early detection of severe liver diseases related to HBV is crucial for the effective treatment. This work aims to investigate the secreted proteins in our recently established cell-based HBV replication system, using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled 2D LC–MS/MS proteomics approach. Such proteins are reflective of early events of HBV infection and thus may have potential as prognostic biomarkers for development of liver diseases.