Hydrogen/Deuterium Exchange Aiding Metabolite Identification in Single-Cell Nanospray High-Resolution Mass Spectrometry Analysis.

The identification of metabolites in single-cell or small-volume tissue samples using single-cell mass spectrometry (MS) is challenging. In this study, hydrogen/deuterium (H/D) exchange was combined with microsampling nanospray high-resolution mass spectrometry (HRMS) to improve the efficiency and confidence level of metabolite identification in a single cell using commercial software. A nanospray ion source showed an improved reaction depth of 8% for H/D exchange compared with an electrospray ion source. In total, 273 metabolites were identified in Allium cepa L. single cells by searching commercial databases. Generally, more than one candidate is given for a precursor ion by MS or tandem MS (MS2) databases such as ChemSpider, MetDNA, MassBank, and mzCloud. With the help of the H/D exchange technique, the number of candidates decreased and reduction of the search space by a factor of 8 was achieved. In addition, two enzymolysis products of isoalliin, the transient intermediate and its isomer, were tracked at the single-cell level using the proposed method.

Rapid Identification of Unknown Organic Iodine in Small-Volume Complex Biological Samples Based on Nanospray Mass Spectrometry Coupled with in-Tube Solid Phase Microextraction.

A new method for rapid screening of unknown organic iodine (OI) in small-volume complex biological samples was developed using in-tube solid phase microextraction (SPME) nanospray mass spectrometry (MS). The method proposed a new identification scheme for OI based on nanospray high-resolution mass spectrometry (HR-MS). The mass ranges of OI ions were confirmed using the t-MS2 scan mode first; then, the possible precursor ions of OI were selected and identified orderly in full MS/ddMS2 and t-MS2 scan modes. Besides, in-tube SPME was used for the pretreatment of small-volume biological samples, and it was the first time in-tube SPME combined with nanospray MS for OI identification. The whole analysis procedure took only 8 min and consumed 50 µL per sample. Using the new method, six kinds of OI added to urine and an unknown OI C12H23O11I in human milk were successfully identified. Moreover, the proposed identification scheme is also suitable for other ambient mass spectrometry (AMS) to determine unknown compounds with characteristic fragment ions.

Selective Identification of Organic Iodine Compounds Using Liquid Chromatography-High Resolution Mass Spectrometry.

A method to selectively and sensitively detect organic iodine compounds and identify their structures has been developed using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Using extracted ion chromatograms of product ions (iodine ion) collected on a rapid scanning quadrupole orbitrap mass spectrometer, the retention times of the unknown organic iodine compounds were determined, and the structural information were acquired according to the MS/MS experiments and the matching with reference standards. We have demonstrated the application of this method by identifying unknown organic iodine compounds in seaweed. A total of 28 possible organic iodine peaks were discovered, among them, the accurate mass and element composition of the corresponding precursor ions were identified for 12 peaks, and molecular structures were confirmed for 4 peaks, which were 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, 4-iodophenol, and 2-iodobenzoic acid. This method is expected to lead to the future discovery of new organic iodine compounds via LC-HRMS in different environmental samples, which is crucial for understanding the iodine biogeochemical cycling.

In situ Identification of Labile Precursor Compounds and their Short-lived Intermediates in Plants using in vivo Nanospray High-resolution Mass Spectrometry.

INTRODUCTION: Many secondary metabolites in plants are labile compounds which under environmental stress, are difficult to detect and track due to the lack of rapid in situ identification techniques, making plant metabolomics research difficult. Therefore, developing a reliable analytical method for rapid in situ identification of labile compounds and their short-lived intermediates in plants is of great importance. OBJECTIVE: To develop under atmospheric pressure, a rapid in situ method for effective identification of labile compounds and their short-lived intermediates in fresh plants. METHODOLOGY: An in vivo nanospray high-resolution mass spectrometry (HR-MS) method was used for rapid capture of labile compounds and their short-lived intermediates in plants. A quartz capillary was partially inserted into fresh plant tissues, and the liquid flowed out through the capillary tube owing to the capillary effect. A high direct current (d.c.) voltage was applied to the plant to generate a spray of charged droplets from the tip of the capillary carrying bioactive molecules toward the inlet of mass spectrometer for full-scan and MS/MS analysis. RESULTS: Many labile compounds and short-lived intermediates were identified via this method: including glucosinolates and their short-lived intermediates (existing for only 10 s) in Raphanus sativus roots, alliin and its conversion intermediate (existing for 20 s) in Allium sativum and labile precursor compound chlorogenic acid in Malus pumila Mill. CONCLUSION: The method is an effective approach for in situ identification of internal labile compounds and their short-lived intermediates in fresh plants and it can be used as an auxiliary tool to explore the degradation mechanisms of new labile plant compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Occurrence and source identification of antibiotics and antibiotic resistance genes in groundwater surrounding urban hospitals.

Urban groundwater, serving as a critical reservoir for potable water, faces susceptibility to contamination from discrete sources such as hospital wastewater. This study investigates the distribution and plausible origins of antibiotics and antibiotic resistance genes (ARGs) in urban groundwater, drawing comparisons between areas proximal to hospitals and non-hospital areas. Ofloxacin and oxytetracycline emerged as the prevalent antibiotics across all samples, with a discernibly richer array of antibiotic types observed in groundwater sourced from hospital-adjacent regions. Employing a suite of multi-indicator tracers encompassing indicator drugs, Enterococci, ammonia, and Cl/Br mass ratio, discernible pollution from hospital or domestic sewage leakage was identified in specific wells, correlating with an escalating trajectory in antibiotic contamination. Redundancy analysis underscored temperature and dissolved organic carbon as principal environmental factors influencing antibiotics distribution in groundwater. Network analysis elucidated the facilitating role of mobile genetic elements, such as int1 and tnpA-02 in propagating ARGs. Furthermore, ARGs abundance exhibited positive correlations with temperature, pH and metallic constituents (e.g., Cu, Pb, Mn and Fe) (p < 0.05). Notably, no conspicuous correlation manifested between antibiotics and ARGs. These findings accentuate the imperative of recognizing the peril posed by antibiotic contamination in groundwater proximal to hospitals and advocate for the formulation of robust prevention and control strategies to mitigate the dissemination of antibiotics and ARGs.

Identification and Localization of Labile Molecules in Broccoli Cell by CLA-ICP-MS and Single-Cell Nanospray HRMS.

Although molecular analysis and imaging by mass spectrometry are emerging as tools to identify metabolites and determine their distribution in cells and tissues, it is difficult to directly analyze the labile molecules at the single-cell level. Glucosinolate (GL) is a plant-active substance with much attention as a chemical defense mechanism known as a "mustard oil bomb" in broccoli. When tissue is damaged, these substances undergo rapid degradation, making them unsuitable for conventional mass spectrometry (MS), particularly for surface MS imaging analysis methods that necessitate intricate preprocessing. Herein, a strategy combining cryogenic laser ablation inductively coupled mass spectrometry (CLA-ICP-MS) and capillary microsampling nanospray high-resolution mass spectrometry (HRMS) was developed. The sulfur-rich microzone in tissue which was thought as a suspect GL-rich cell population was located via CLA-ICP-MS. Three GLs in single cells were accurately identified by nanospray HRMS with a hydrogen/deuterium exchange reaction. Subsequently, cell-by-cell imaging by nanospray HRMS showed that the GL-rich cells were below the stalk surface by approximately 30 µm. This proposed strategy can also be applied to rapidly identify labile compounds and localize molecule-rich cells in tissues.

In vivo nanoelectrospray for the localization of bioactive molecules in plants by mass spectrometry.

The method for the localization of bioactive molecules in plants is highly needed since it provides a fundamental prerequisite for understanding their physiological and ecological functions. Here, we propose a simple method termed in vivo nanoelectrospray for the localization of bioactive molecules in plants without sample preparation. A capillary is partly inserted into the plant to sample liquid from a highly located region, and then, a high voltage is applied to the plant to generate an electrospray from the capillary tip for mass spectrometry analysis. Using this method, bioactive molecules such as saccharides, glycoalkaloids, flavonoids, organic acids, and glucosinolates (GLs) are detected in the target regions of living plants or fresh fruits. Original information for endogenous chemicals including liable molecules in plant can be obtained. A sketchy three-dimensional distribution of glycoalkaloids in a cherry tomato has been obtained. The present work provides a powerful tool for the study of bioactive molecules in a living plant by mass spectrometry.

Determination of As species distribution and variation with time in extracted groundwater samples by on-site species separation method.

It is challenging to dependably keep the native distribution of arsenic (As) species before sample analysis in the laboratory. The on-site separation method can avoid sample contamination and species change in the process of sample collection and transportation from field to laboratory. In this study, As species distribution and variation of the extracted groundwater was first analyzed by an on-site species separation method in Jianghan Plain, China. Our study illustrated that: 1) high-As groundwater generally existed under mildly reducing conditions (Eh < 200 mV), weak alkaline conditions (pH < 7.2), elevated concentrations of dissolved Fe(II) and S(-II), and high proportions of As (III); 2) As species in the groundwater changed dramatically at room temperature in 36 hours post extraction (HPE). Fe-sulfide and Fe oxides minerals, which adsorbed As (V), were the main reasons influencing the As species concentration; 3) Acidification and strong complexing agents cannot preserve As species effectively. The average proportion of As (III) in the wells, where groundwater samples from the depth of 25 m exceed 10 µg L-1 As, can be reduced by 61% and 63% after HCl and EDTA were added, respectively. Accurate assessment of concentrations and distribution variation of As species in groundwater can guide the removal of As and the safe use of water resources, especially in drought areas relying on drinking well water.

Controlling charge states of peptides through inductive electrospray ionization mass spectrometry.

A novel ionization device for controlling the charge states of peptides based on an inductive elecrospray ionization technique was developed. This ion source keeps the major capabilities of electrospray ionization (ESI) which is compatible with liquid separation techniques (such as liquid chromatography (LC) and capillary electrophoresis (CE)) and can be potentially used to control the charge states of peptides accurately by simply varying the AC voltage applied. In comparison with conventional ESI, inductive ESI successfully simplifies the mass spectrum by reducing the charge states of peptide to a singly charged one, as well as eliminating the adduct ions.

The identification of halogenated disinfection by-products in tap water using liquid chromatography-high resolution mass spectrometry.

In this paper, a comprehensive method for the identification of the unknown halogenated DBPs (X-DBPs, X = Cl, Br, and I) in the tap water of Wuhan, China via liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed. 123 X-DBPs were identified through the stepwise procedure, 94 of them were newly identified, and 3 of them were confirmed by standards. Most X-DBPs were aliphatic compounds and highly unsaturated and phenolic compounds, some X-DBPs contained multiple halogen atoms and rich in carboxyl groups, such as C2H2O2BrCl, C2H2O2Br2, and C2H2O2ClI. It was worth noting that the concentration of some X-DBPs had the same trend with time. Most Cl-DBPs remained stable and I-DBPs were detected occasionally by monitoring the change of concentration of these X-DPBs with the time during three consecutive months. The results demonstrate that the proposed method could provide valuable molecular formula and structure information on unknown multiple halogenated DBPs, or be used for the identification of other multiple halogenated organic compounds in different media.

Characterization of the diethyl phthalate-degrading bacterium Sphingobium yanoikuyae SHJ.

A newly isolated bacterial strain SHJ was found to be capable of degrading diethyl phthalate (DEP) very efficiently. Its growth characteristics and 16S rDNA gene sequence were analyzed. Its whole genome was also sequenced. Strain SHJ was identified as Sphingobium yanoikuyae SHJ.

Suspect screening and target quantification of human pharmaceutical residues in the surface water of Wuhan, China, using UHPLC-Q-Orbitrap HRMS.

In this study we developed a systematic method for suspect screening and target quantification of the human pharmaceutical residues in water, via solid phase extraction (SPE) followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). We then proceeded to study the occurrences and distribution of the pharmaceuticals in the surface waters of Wuhan, China, by analyzing water samples from lakes, rivers and municipal sewage. Initially, 33 human pharmaceuticals were identified from East Lake without using purchasing standards. Of these, 29 were later confirmed by using standards, and quantified using the aforementioned SPE pretreatment method and LC-HRMS analysis in full MS scan mode. The 29 compounds included 8 antibiotics, 9 metabolites, and 12 miscellaneous pharmaceuticals. The highest proportions of pharmaceutical residues were detected downstream of the Yangtze River and in the lakes close to the central city. Metformin, cotinine, and trans-3-hydroxy cotinine, were frequently encountered in all the surface water samples. High concentrations (>120 ng/l) of caffeine, metformin, theobromine, and valsartan were detected in the surface water samples; the removal rates of these compounds in the municipal sewage treatment plant were also high. In contrast, although the concentrations of 4-AAA and metoprolol acid in the surface water were high, the removal rates of these residues in the sewage treatment plant were low.

A Pck1-directed glycogen metabolic program regulates formation and maintenance of memory CD8+ T cells.

CD8+ memory T (Tm) cells are fundamental for protective immunity against infections and cancers 1-5 . Metabolic activities are crucial in controlling memory T-cell homeostasis, but mechanisms linking metabolic signals to memory formation and survival remain elusive. Here we show that CD8+ Tm cells markedly upregulate cytosolic phosphoenolpyruvate carboxykinase (Pck1), the hub molecule regulating glycolysis, tricarboxylic acid cycle and gluconeogenesis, to increase glycogenesis via gluconeogenesis. The resultant glycogen is then channelled to glycogenolysis to generate glucose-6-phosphate and the subsequent pentose phosphate pathway (PPP) that generates abundant NADPH, ensuring high levels of reduced glutathione in Tm cells. Abrogation of Pck1-glycogen-PPP decreases GSH/GSSG ratios and increases levels of reactive oxygen species (ROS), leading to impairment of CD8+ Tm formation and maintenance. Importantly, this metabolic regulatory mechanism could be readily translated into more efficient T-cell immunotherapy in mouse tumour models.

Identification of Organic Iodine Compounds and Their Transformation Products in Edible Iodized Salt Using Liquid Chromatography-High Resolution Mass Spectrometry.

The consumption of edible iodized salt is a key strategy to control and eliminate iodine deficiency disorders worldwide. We herein report the identification of the organic iodine compounds present in different edible iodized salt products using liquid chromatography combined with high resolution mass spectrometry. A total of 38 organic iodine compounds and their transformation products (TPs) were identified in seaweed iodine salt from China. Our experiments confirmed that the TPs were generated by the replacement of I atoms from organic iodine compounds with Cl atoms. Furthermore, the organic iodine compound contents in 4 seaweed iodine salt samples obtained from different manufacturers were measured, with significant differences in content being observed. We expect that the identification of organic iodine compounds in salt will be important for estimating the validity and safety of edible iodized salt products.

Thioarsenate Formation Coupled with Anaerobic Arsenite Oxidation by a Sulfate-Reducing Bacterium Isolated from a Hot Spring.

Thioarsenates are common arsenic species in sulfidic geothermal waters, yet little is known about their biogeochemical traits. In the present study, a novel sulfate-reducing bacterial strain Desulfotomaculum TC-1 was isolated from a sulfidic hot spring in Tengchong geothermal area, Yunnan Province, China. The arxA gene, encoding anaerobic arsenite oxidase, was successfully amplified from the genome of strain TC-1, indicating it has a potential ability to oxidize arsenite under anaerobic condition. In anaerobic arsenite oxidation experiments inoculated with strain TC-1, a small amount of arsenate was detected in the beginning but became undetectable over longer time. Thioarsenates (AsO4-xSx2- with x = 1-4) formed with mono-, di- and tri-thioarsenates being dominant forms. Tetrathioarsenate was only detectable at the end of the experiment. These results suggest that thermophilic microbes might be involved in the formation of thioarsenates and provide a possible explanation for the widespread distribution of thioarsenates in terrestrial geothermal environments.

Rapid in situ identification of bioactive compounds in plants by in vivo nanospray high-resolution mass spectrometry.

A method for the rapid in situ identification of bioactive compounds in fresh plants has been developed using in vivo nanospray coupled to high-resolution mass spectrometry (HR-MS). Using a homemade in vivo nanospray ion source, the plant liquid was drawn out from a target region and ionized in situ. The ionized bioactive compounds were then identified using Q-Orbitrap HR-MS. The accurate mass measurements of these bioactive compounds were performed by full-scan or selected ion monitoring (SIM), and tandem mass spectrometry (MS/MS) was used in the structural elucidation. Without sample pretreatment, 12 bioactive compounds in 7 different plant species were identified, namely, isoalliin in onion; butylphthalide in celery; N-methylpelletierine, pelletierine, and pseudopelletierine in pomegranate; chlorogenic acid in crabapple; solamargine, solasonine, and solasodine in nightshade; aloin and aloe-emodin in aloe; and menthone in mint. This work demonstrates that in vivo nanospray HR-MS is a good method for rapid in situ identification of bioactive compounds in plants.

Low-temperature plasma ionization source for the online detection of indoor volatile organic compounds.

A simple-structure, low-power, and low-cost low temperature plasma (LTP) ionization source, coupled with mass spectrometry, for the online detection of indoor volatile organic compounds (VOCs) has been constructed in this work. Air, instead of noble gases, was employed as the discharging and carrier gas. And a custom-built AC high-voltage power supply with a total power consumption of 5 W, frequency of 2-4 kHz, and amplitude around 1-5 kV(p-p) was used. This LTP source is a soft ionization source. The initial performance of the ionization source has been evaluated by ionizing samples including alcohols, ketones, aldehydes and aromatics. These compounds cover most of the common air pollutants concerning people's health. It is well known that those plasmas generated by dielectric barrier discharge (DBD) produce significant amount of metastable species and electrons with mean energies greater than several electronvolt, but minimal fragmentation was observed in our work. Protonated ions are the dominant product for the VOCs detected after the ionization process. Further work has been conducted to confirm the detection feature of this source. The results are promising enough to ensure the novel LTP ionization source as an effective tool for the online detection of indoor VOCs.