Chromatography Fourier Transform Ion Cyclotron Research Articles

Anaerobic degradation of excess protein-rich fish feed drives CH4 ebullition in a freshwater aquaculture pond

Aquaculture is a climate-relevant source of greenhouse gases like methane. Methane emissions depend on various parameters, with organic matter playing a crucial role. Nevertheless, little is known about the composition of organic matter in aquaculture. We investigated the effects of excessive loading of high-protein fish feed on the quality of sediment organic matter in a fishpond to explain extremely high methane ebullition rates (bubble flux). Analysing the molecular composition of water-extractable organic matter using liquid chromatography Fourier-transform ion cyclotron resonance mass spectroscopy, we found strong differences between the feeding area and open water area: low-molecular weight nitrogen and sulphur-rich organic compounds were highly enriched at the feeding area. In addition, methane ebullition correlated well with sediment protein content and total bound nitrogen in pore water. Our results indicate that feed proteins in the sediments are hydrolysed into oligopeptides (CHNO) and subsequently converted to CHOS and CHNOS components during anaerobic deamination of protein and peptide fragments in the presence of inorganic sulphides. These metabolites accumulate at the feeding area due to continuous feed supply. Our findings illustrate the adverse effects of excessive feeding leading to bioreactor-like methane emissions at the feeding area. Improving feed management has the potential to make aquaculture more climate-friendly.

A comprehensive quality evaluation strategy of Shensong Yangxin capsules based on qualitative, fingerprint and quantitative analyses.

Shensong Yangxin capsule (SSYXC), an effective Chinese patent medicine, has been recorded in the Chinese Pharmacopeia, mainly for the treatment of coronary heart disease and ventricular premature beat. To further complete the quality evaluation of SSYXC, a comprehensive analysis strategy was established. Firstly, the components of SSYXC were qualitatively analysed using ultra-high- performance liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. A total of 134 compounds were identified or tentatively characterized. Additionally, the fingerprint of SSYXC was established by HPLC, and the similarity of 10 batches of SSYXC was elucidated by similarity analysis. The result indicated that the consistency of chemical composition is good. Finally, to enhance the quality control of SSYXC, according to the results of the fingerprint analysis, the contents of the seven active components was determined, comprising morroniside, loganin, paeoniflorin, salvianolic acid B, palmatine hydrochloride, berberine hydrochloride and tanshinone IIA. In conclusion, the established method, comprising identification of components, fingerprint analysis and quantification of multicomponents, can be sensitively and comprehensively applied to the quality evaluation of SSYXC, which can provide chemical ingredients bases for quality control and the pharmacodynamic mechanism of SSYXC, which could serve as a benchmark for controlling the quality of other Chinese patent medicines.

Chemical diversity of dissolved organic matter revealed by ultra performance liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry

Dissolved organic matter (DOM) is a highly complex and heterogeneous mixture that exists in various environments, including rivers, oceans, soils, and atmospheric aerosols. DOM plays a crucial role in biogeochemical cycles and significantly influences the environment by regulating water quality, changing the climate, and transporting pollutants. Therefore, clarifying the detailed molecular composition of DOM is essential to obtain a better understanding of its physical and chemical properties, thereby enabling further elucidation of its biogeochemical behavior. In this study, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with quadrupole detection (QPD) was used to conduct the online ultra performance liquid chromatography (UPLC)-MS analysis of DOM in water, aerosol, and soil samples collected in Tianjin, China. The samples were extracted with pure water and filtered through a glass fiber membrane (0.45 μm). The DOM in the samples was then enriched by solid-phase extraction (SPE) and redissolved in water-acetonitrile (1∶1, v/v) at mass concentration of 200 mg/L for the LC-MS experiments. The mobile phases used for UPLC were water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient elution procedure was as follows: 0-5 min, 0B; 5-11 min, 0B-95%B; 11-25 min, 95%B; 25-28 min, 95%B-0B; 28-30 min, 0B. The flow rate was 0.1 mL/min, and the injection volume was 10 μL. The UV wavelength was set at 274 nm. MS detection was performed in negative electrospray ionization (ESI(-)) mode with a capillary voltage of 5.0 kV, and the MS data were collected in broadband (m/z 150-1000) and QPD modes. The transient data size was set to 2M, the free induction decay signal length was 0.74 s, and the ion accumulation time was 0.030 s. Four chromatographic peaks were observed in the chromatograms. The first peak was identified as salt adduct compounds containing sodium formate. The three other peaks contained complex components, such as oxygen-rich, unsaturated tannin-like compounds, as well as low-oxygen, highly saturated lignin-like and protein/amino-like compounds. UPLC-FT-ICR MS was suitable for assigning the detailed elemental compositions of the DOM samples. UPLC effectively improved the ionization efficiency of difficult-to-ionize compounds and enhanced the detection accuracy of MS. Indeed, MS peaks with a mass difference of as small as 3.4 mDa were well identified. A total of 12027, 15593, and 8029 peaks in the mass spectra of the water, aerosol, and soil samples, respectively, were assigned to known elemental formulae. Peaks Ⅱ and Ⅲ were hydrophilic components mainly including CHNO and CHO compounds. Compared with peak Ⅱ, peak Ⅲ exhibited a significant increase in CHNOS and CHOS, indicating that UPLC exerted a certain separation effect on these compounds. Furthermore, the aerosol samples contained a higher concentration of sulfur-containing compounds than the water and soil samples, primarily because of the abundance of organic sulfates present in atmospheric and cloud water. Data processing and graphic visualization revealed that the unique components in the water samples mainly appeared in the area of 0.1<O/C<0.5 and 1.0 <H/C<1.7. The compounds detected were low-oxygen and highly condensed lignin-like compounds. The unique components in the aerosol samples appeared in the area of 0.4<O/C<1.0 and 1.5<H/C<2.0, and were classified as carbohydrates. The unique components in the hydrophilic fraction of the soil samples were found in the area of 0.6<O/C<1.0 and 0.5<H/C<1.0, and were determined to be tannin-like compounds. By contrast, the components in the hydrophobic fraction were similar to those found in the water samples and appeared in the region containing lignin-like compounds. In summary, this study proposed a novel analytical protocol to characterize DOM from different ecosystems using UPLC-FT-ICR MS. This method could separate DOM components using UPLC with eluents of different polarities and analyze them using high-resolution FT-ICR MS to reveal their molecular compositions and possible chemical types. This protocol offers solid technical support for the comprehensive profiling of DOM at the molecular level.

Rapid characterization of the chemical constituents of Duzhong Jiangya tablet by HPLC coupled with Fourier transform ion cyclotron resonance mass spectrometry.

Duzhong Jiangya tablet is a hypotensive drug. In this study, high-performance liquid chromatography-Fourier transform-ion cyclotron resonance-mass spectrometry technology was used to quickly identify its chemical composition. SinoChrom ODS-BP column (250mm × 4.6mm, 5 μm) was used. The mobile phase was acetonitrile(A)-0.1% formic acid solution(B). The flow rate was 1mL/min. Extracted ion chromatogram was used to analyze the samples in positive and negative ion modes. Based on the accurate mass spectrometry information (such as quasi-molecular ions and fragment ions) obtained from the instrument, combined with reference compounds and literature, the chemical composition of Duzhong Jiangya Tablets was identified. A total of 131 compounds were identified, including four types of penylpropanoids, six types of phenylethanoid glycosides, 10 types of organic acids, 14 types of iridoids, 12 types of lignans, 18 types of alkaloids, seven types of coumarins, and 60 kinds of flavonoids. This established method can quickly and efficiently identify chemical constituents in Duzhong Jiangya tablet, lay a foundation for the research on the efficacy and quality of this traditional Chinese medicine, and provide a reference for the characterization of the chemical constituents of other traditional Chinese medicine preparations.

Metabolome profile of Negi-Nira chive, an interspecies hybrid of green spring onion (Allium fistulosum) and Chinese chive (A. tuberosum).

Metabolome analysis of flavored vegetables, green spring onion (Allium fistulosum), Chinese chive (A. tuberosum), and their interspecies hybrid Negi-Nira chive, was conducted using liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry, with ca. 2 ppm mass accuracy. Ion peaks in the chromatograms of four biological replicates of the vegetable leaves were processed using the alignment software PowerGet for metabolite comparison, from which we obtained the potential chemical formulae. In total, 860 ion peaks were reproducibly detected; of these, 506, 525, and 336 peaks were found in the hybrid, A. tuberosum, and A. fistulosum, respectively. There were 130 peaks specific to the hybrid; from these, 31 metabolites were annotated by searching compound databases. The sulfur-containing compounds and flavonoids were further analyzed using bioinformatics, to examine the sulfur metabolism of Allium volatiles and the flavonoid pathways in these species. In conclusion, our metabolome analysis of this interspecies hybrid and its parents provides a unique opportunity to elucidate their metabolic background.

Metabolic reprogramming of hydrogenosomal amino acids in Trichomonas vaginalis under glucose restriction.

Glucose is the major energy source that is converted to pyruvate for ATP generation in the trichomonad hydrogenosome. Under glucose restriction (GR), the regulation of amino acids metabolism is crucial for trichomonad growth and survival. RNA-sequencing (RNA-seq) analysis has been used to identify differentially expressed genes in Trichomonas vaginalis under GR, leading to significant advances in understanding adaptive responses of amino acid metabolism to GR. However, the levels of amino acid metabolites modulated by GR are unknown in T. vaginalis. Herein, we describe a comprehensive metabolomic analysis of amino acid metabolites in the hydrogenosome using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FT MS). The relative abundance of 17 hydrogenosomal amino acids was analyzed under GR and high-glucose (HG) conditions. Levels of most amino acids were higher in GR culture. Arginine was not detectable in either HG or GR cultures; however, its metabolic end-product proline was slightly increased under GR, suggesting that the arginine dihydrolase pathway was more activated by GR. Additionally, methionine catabolism was less stimulated under GR because of greater methionine accumulation. Furthermore, branched chain amino acids (BCAA), including leucine, isoleucine and valine, as well as phenylalanine and alanine, markedly accumulated under GR, indicating that glutamate-related metabolic pathways were remarkably enhanced in this setting. Our metabolomic analysis combined with previous RNA-seq data confirm the existence of several amino acid metabolic pathways in the hydrogenosome and highlight their potentially important roles in T. vaginalis under glucose deprivation.

Human Dendritic Cells with Th2-Polarizing Capacity: Analysis Using Label-Free Quantitative Proteomics

Background: Dendritic cells (DCs) are the sentinels of the immune system. Upon recognition of a pathogen, they mature and migrate to draining lymph nodes to prime and polarize T cell responses. Although it is known that helminths and helminth-derived molecules condition DCs to polarize T helper (Th) cells towards Th2, the underlying mechanisms remain incompletely understood. Objectives: The aim of this study was to conduct a proteome analysis of helminth antigen-stimulated DCs in order to gain more insight into the cellular processes associated with their ability to polarize immune responses. Methods: We analyzed the maturation and polarization of monocyte-derived DCs from 9 donors at 2 different time points after stimulation with different Th1- and Th2-polarizing pathogen-derived molecules. The samples were measured using liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for relative quantitation. Results: Lipopolysaccharide-induced maturation promoted the expression of proteins related to metabolic, cellular, and immune system processes. Th1-polarizing DCs, conditioned by IFN-γ during maturation, displayed accelerated maturation by differentially expressing cytoskeletal proteins and proteins involved in immune regulation. The stimulation of DCs with soluble egg antigens and omega-1 derived from Schistosoma mansoni, which are both Th2-inducing stimuli, increased 60S acidic ribosomal protein P2, and vesicle amine transferase 1 while decreasing the expression of proteins related to antigen processing and presentation. Conclusion: Our data indicate that not only proteins involved in the interaction between T cells and DCs at the level of the immunological synapse, but also those related to cellular metabolism and stress, may promote Th2 polarization.

Evidence for High Concentrations and Maternal Transfer of Substituted Diphenylamines in European Eels Analyzed by Two-Dimensional Gas Chromatography–Time-of-Flight Mass Spectrometry and Gas Chromatography–Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Chemical pollution is hypothesized to be one of the factors driving the strong decline of the critically endangered European eel population. Specifically, the impact of contaminants on the quality of spawning eels and subsequent embryo survival and development has been discussed as crucial investigation point. However, so far, only very limited information on potential negative effects of contaminants on the reproduction of eels is available. Through the combination of nontargeted ultrahigh-resolution mass spectrometry and multidimensional gas chromatography, combined with more-conventional targeted analytical approaches and multimedia mass-balance modeling, compounds of particular relevance, and their maternal transfer in artificially matured European eels from the German river Ems have been identified. Substituted diphenylamines were, unexpectedly, found to be the primary organic contaminants in the eel samples, with concentrations in the μg g-1 wet weight range. Furthermore, it could be shown that these contaminants, as well as polychlorinated biphenyls (PCBs), organochlorine pesticides, and polyaromatic hydrocarbons (PAHs), are not merely stored in lipid rich tissue of eels but maternally transferred into gonads and eggs. The results of this study provide unique information on both the fate and behavior of substituted diphenylamines in the environment as well as their relevance as contaminants in European eels.

Wolbachia Modulates Lipid Metabolism in Aedes albopictus Mosquito Cells.

ABSTRACTCertain strains of the intracellular endosymbiont Wolbachia can strongly inhibit or block the transmission of viruses such as dengue virus (DENV) by Aedes mosquitoes, and the mechanisms responsible are still not well understood. Direct infusion and liquid chromatography-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry-based lipidomics analyses were conducted using Aedes albopictus Aa23 cells that were infected with the wMel and wMelPop strains of Wolbachia in comparison to uninfected Aa23-T cells. Substantial shifts in the cellular lipid profile were apparent in the presence of Wolbachia. Most significantly, almost all sphingolipid classes were depleted, and some reductions in diacylglycerols and phosphatidylcholines were also observed. These lipid classes have previously been shown to be selectively enriched in DENV-infected mosquito cells, suggesting that Wolbachia may produce a cellular lipid environment that is antagonistic to viral replication. The data improve our understanding of the intracellular interactions between Wolbachia and mosquitoes.IMPORTANCE Mosquitoes transmit a variety of important viruses to humans, such as dengue virus and Zika virus. Certain strains of the intracellular bacterial genus called Wolbachia found in or introduced into mosquitoes can block the transmission of viruses, including dengue virus, but the mechanisms responsible are not well understood. We found substantial shifts in the cellular lipid profiles in the presence of these bacteria. Some lipid classes previously shown to be enriched in dengue virus-infected mosquito cells were depleted in the presence of Wolbachia, suggesting that Wolbachia may produce a cellular lipid environment that inhibits mosquito-borne viruses.

Boosting Sensitivity in Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance-Tandem Mass Spectrometry for Product Ion Analysis of Monoterpene Indole Alkaloids.

In metabolomics, the analysis of product ions in tandem mass spectrometry (MS/MS) is noteworthy to chemically assign structural information. However, the development of relevant analytical methods are less advanced. Here, we developed a method to boost sensitivity in liquid chromatography–Fourier transform ion cyclotron resonance–tandem mass spectrometry analysis (MS/MS boost analysis). To verify the MS/MS boost analysis, both quercetin and uniformly labeled 13C quercetin were analyzed, revealing that the origin of the product ions is not the instrument, but the analyzed compounds resulting in sensitive product ions. Next, we applied this method to the analysis of monoterpene indole alkaloids (MIAs). The comparative analyses of MIAs having indole basic skeleton (ajmalicine, catharanthine, hirsuteine, and hirsutine) and oxindole skeleton (formosanine, isoformosanine, pteropodine, isopteropodine, rhynchophylline, isorhynchophylline, and mitraphylline) identified 86 and 73 common monoisotopic ions, respectively. The comparative analyses of the three pairs of stereoisomers showed more than 170 common monoisotopic ions in each pair. This method was also applied to the targeted analysis of MIAs in Catharanthus roseus and Uncaria rhynchophylla to profile indole and oxindole compounds using the product ions. This analysis is suitable for chemically assigning features of the metabolite groups, which contributes to targeted metabolome analysis.

Using community metabolomics as a new approach to discriminate marine microbial particulate organic matter in the western English Channel

Metabolomics provides an unbiased assessment of a wide range of metabolites and is an emerging ‘omics technique in the marine sciences. We use ‘non-targeted’ community metabolomics to determine patterns in metabolite profiles associated with particulate organic matter (POM) at four locations from two long-term monitoring stations (L4 and E1) in the western English Channel. The polar metabolite fractions were measured using ultra-high performance liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS), and the lipid fractions by direct infusion Fourier transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS); these were then analysed to statistically compare the metabolite distributions. Results show significantly different profiles of metabolites across the four locations with the largest differences for both the polar and lipid fractions found between the two stations relative to the smaller differences associated with depth. We putatively annotate the most discriminant metabolites revealing a range of amino-acid derivatives, diacylglyceryltrimethylhomoserine (DGTS) lipids, oxidised fatty acids (oxylipins), glycosylated compounds, oligohexoses, phospholipids, triacylglycerides (TAGs) and oxidised TAGs. The majority of the polar metabolites were most abundant in the surface waters at L4 and least abundant in the deep waters at E1 (E1-70m). In contrast, the oxidised TAGs were more abundant at E1 and most abundant at E1-70m. The differentiated metabolites are discussed in relation to the health of the phytoplankton as indicated by nutrients, carbon and chlorophyll, and to the dominance (determined from metatranscript data) of the picoeukaryote Ostreococcus. Our results show proof of concept for community metabolomics in discriminating and characterising polar and lipid metabolite patterns associated with marine POM.

Characterization of naphthenic acids by gas chromatography-Fourier transform ion cyclotron resonance mass spectrometry.

During the bitumen extraction from the oil sands of Alberta, large volumes of process water containing naphthenic acids are stored in tailing ponds. The naphthenic acids along with other components in the processed waters are known to be toxic in aquatic environments. In view of the complex matrix and the toxicity of the processed waters, there is a need for complementary analytical techniques for comprehensive characterization of the naphthenic acid mixtures. This study reports the online gas chromatographic separation of naphthenic acid mixtures prior to ultrahigh resolution mass spectrometry detection, using electron and chemical ionization. Two oil sands processed water samples and two groundwater samples were characterized to evaluate the performance of the instrumental technique. The high mass resolution of the system enabled visualization of the data using Kendrick mass defect plots. The addition of gas chromatographic separations enabled visualization of the data as unique compound class elution fingerprints. The technique is demonstrated to be a valuable tool for chemical fingerprinting of naphthenic acids.

Metabolomic profiling of bronchoalveolar lavage fluids by isotope labeling liquid chromatography mass spectrometry: a promising approach to studying experimental asthma

Metabolomic analysis of bronchoalveolar lavage fluid (BALF) may help understand the pathophysiology of pulmonary diseases such as asthma. However, a major analytical challenge is that most metabolites in BALF are in low abundance and further diluted by the collection procedure. Here we report a sensitive metabolomic profiling method based on 12C-/13C-differential isotope dansylation that targets amine- and phenol-containing metabolites, solid phase extraction for analyte enrichment, and liquid chromatography Fourier transform ion cyclotron resonance-mass spectrometry (LC–FTICR-MS). This method detected 250 ion pairs or putative metabolites in rat BALF and 36 of them were positively identified. The majority were not reported in previous studies using NMR or conventional LC–MS. The developed isotope labeling method was then applied to investigate metabolomic changes in BALF samples from a model of allergic asthma in rats. Statistical analysis of the resultant data showed that there was distinct separation between normal and inflamed rats. Metabolic pathway analysis indicated that the arginine-proline metabolic pathway was dysregulated in rats with experimental asthma.

Characterization of Highly Oxygenated and Environmentally Weathered Crude Oils by Liquid Chromatography Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR MS)

Heavy and biodegraded oils exhibit an ever increasing oxygen complexity. Electrospray ionization (ESI) coupled to FT-ICR MS highlights the most acidic and basic oxygen species; however, quantitative and qualitative information about less polar oxygenated compounds is essential to understand both the source and, potentially, the modification pathways for oxygenated crude oils. Liquid chromatography (LC) is complementary to FT-ICR MS to deconvolve crude oils according to oxygen functionality, thereby enriching oxygen species that are not easily ionized or are present in low abundance. Ketones and carboxylic acids dominate most environmentally modified crude oils; however, phenols and polyphenols are also important intermediates in both biotic and abiotic modifications. Moreover, due to the nature of the refinement process, oxygen-containing species and their intermediates can serve as chemical tracers in produced water streams and areas of rapid thermal changes that lead to gum or deposit formation. Here, we describe an analytical separation scheme for an environmentally weathered (highly oxygenated) crude oil designed to highlight carboxylic acid isolation and ketone enrichment, as well as the separation of other oxygen compounds by polarity. Experimental A weathered beach extract collected from the coast of Florida was fractionated by LC and analyzed by FT-ICR MS. LC techniques are designed to target specific oxygen functionalities (e.g., ketones, alcohols, phenols, and carboxylic acids). The mass spectra of the LC fractions were acquired with a custom-built 9.4 T FT-ICR Instrument. Positive and negative electrospray ionization serve to highlight basic and acidic oxygen species. Preliminary DataLC fractionation was performed with a silicic acid stationary phase. Four fractions correspond to aromatics, neutral nitrogen, basic nitrogen, and polar compounds. Positive ESI FT-ICR MS indicated a relative abundance for the O1 heteroatom class ranging from 2% in the whole beach extract to 12 % in the neutral nitrogen fraction. The increased relative abundance of O1 compounds suggests the enrichment of ketones from the original sample. Carboxylic acids are isolated by retention on an amine stationary phase and subsequent elution with a mixture of dichloromethane, methanol, and formic acid. Oxygen species will be further probed with new separation schemes to focus on analysis of phenols.

5-Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry

We describe a new set of isotope reagents, (12)C4-, (12)C2(13)C2-, and (13)C4-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl).

Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The 12C-/13C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10μL of urine. We believe that this method opens the possibility of generating a very comprehensive profile of the organic acid sub-metabolome that will be useful for comparative metabolomics applications for biological studies and disease biomarker discovery.

Combination of Liquid Chromatography–Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry with 13C-Labeling for Chemical Assignment of Sulfur-Containing Metabolites in Onion Bulbs

Phytochemicals containing heteroatoms (N, O, S, and halogens) often have biological activities that are beneficial to humans. Although targeted profiling methods for such phytochemicals are expected to contribute to rapid chemical assignments, thus making phytochemical genomics and crop breeding much more efficient, there are few profiling methods for the metabolites. Here, as an ultrahigh performance approach, we propose a practical profiling method for S-containing metabolites (S-omics) using onions (Allium cepa) as a representative species and (12)C- and (13)C-based mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses by liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC-FTICR-MS). Use of the ultrahigh quality data from FTICR-MS enabled simplifying the previous methods to determine specific elemental compositions. MS analysis with a resolution of >250,000 full width at half-maximum and a mass accuracy of <1 ppm can distinguish S-containing monoisotopic ions from other ions on the basis of the natural abundance of (32)S and (34)S and the mass differences among the S isotopes. Comprehensive peak picking using the theoretical mass difference (1.99579 Da) between (32)S-containing monoisotopic ions and their (34)S-substituted counterparts led to the assignment of 67 S-containing monoisotopic ions from the (12)C-based MS spectra, which contained 4693 chromatographic ions. The unambiguous elemental composition of 22 ions was identified through comparative analysis of the (12)C- and (13)C-based MS spectra. Finally, of these, six ions were found to be derived from S-alk(en)ylcysteine sulfoxides and glutathione derivatives. This S-atom-driven approach afforded an efficient chemical assignment of S-containing metabolites, suggesting its potential application for screening not only S but also other heteroatom-containing metabolites in MS-based metabolomics.

Development of Isotope Labeling LC–MS for Human Salivary Metabolomics and Application to Profiling Metabolome Changes Associated with Mild Cognitive Impairment

Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential (13)C/(12)C isotope dansylation labeling method, combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), is described for quantitative profiling of the human salivary metabolome. New strategies are presented to optimize the sample preparation and LC-MS detection processes. The strategies allow the use of as little of 5 μL of saliva sample as a starting material to determine the concentration changes of an average of 1058 ion pairs or putative metabolites in comparative saliva samples. The overall workflow consists of several steps including acetone-induced protein precipitation, (12)C-dansylation labeling of the metabolites, and LC-UV measurement of the total concentration of the labeled metabolites in individual saliva samples. A pooled sample was prepared from all the individual samples and labeled with (13)C-dansylation to serve as a reference. Using this metabolome profiling method, it was found that compatible metabolome results could be obtained after saliva samples were stored in tubes normally used for genetic material collection at room temperature, -20 °C freezer, and -80 °C freezer over a period of 1 month, suggesting that many saliva samples already collected in genomic studies could become a valuable resource for metabolomics studies, although the effect of much longer term of storage remains to be determined. Finally, the developed method was applied for analyzing the metabolome changes of two different groups: normal healthy older adults and comparable older adults with mild cognitive impairment (MCI). Top-ranked 18 metabolites successfully distinguished the two groups, among which seven metabolites were putatively identified while one metabolite, taurine, was definitively identified.

Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila

Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.

Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. (1)H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.