The Exactive mass spectrometer has a maximum scan range of m/z 50−4000. For the study of small molecule metabolites, of particular interest is the low mass range (m/z 85−1000). In preliminary experiments, we found that the high amount of phosphate and sulfate in typical cellular media adversely impact analysis, apparently due to a combination of ion suppression at the ion source and space-charge effects inside the orbitrap. To mitigate the latter, during the LC segments at which phosphate (H2PO4−, m/z 96.9696, ∼6 min) and sulfate (HSO4−, m/z 96.9601, ∼13 min) elute, we chose a lower scan limit of ≥m/z 100 (rather than m/z 85) to reduce the accumulation of phosphate and sulfate ions in the orbitrap. Although the scan limit does not provide high-resolution mass filtration, ions falling outside of the scan range are selected against. Empirically, we found that this selection was adequate to substantially improve the analytical results. Later in the LC run, the lower m/z limit was raised yet higher, as low molecular weight metabolites elute early in this reversed phase LC method. The final MS scan method is thus made in the following with segments: 0−5 min, m/z 85−800; 5−6.7 min, m/z 100−800; 6.7−9 min, m/z 85−800; 9−16 min, m/z 110−1000; 16−24 min, m/z 220−1000. The last minute in the LC run is for column equilibrium only and is not scanned. Other MS method settings are resolution 100 000 at 1 Hz (1 scan per second), AGC (automatic gain control) target 3 × 106, maximum injection time 100 μS. Anal. Chem. 2010, 82, 8, 3212–3221
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse phase"],"chromatography_protocol":["The metabolite extracts were analyzed by reversed-phase ion-pairing liquid chromatography using ultra-high performance LC (Thermo Scientific DionexUltiMate 3000) coupled with high-resolution/accurate-mass spectrometer (Thermo Scientific Q Exactive quadrupole-Orbitrap hybrid mass spectrometer) with electrospray ionization operated in negative mode. An injection sample of 10 μL was used and the column temperature was set to 25°C. A Waters Acquity UPLC BEH C18 1.7 μm with a column size of 2.1 × 100 mm) (Waters Corporation, Massachusetts) was used. Solvent A contained 97:3 (v:v) LC-MS grade H2O: methanol with acetic acid (15 mM) and tributylamine (10 mM). Solvent B contained 100% methanol. The flow rate was 180 μL min−1 during the entire sample run (25 min). The solvent gradient with respect to solvent A was the following: 0 min, 100%; 2.5 min, 100%; 5 min, 80%; 7.5 min, 80%; 10 min, 45%; 12 min, 45%; 14 min, 5%; 17 min, 5%; 18 min, 0%; 25 min, 0% was run.
"],"publication":["Nutrient depletion and systems-wide metabolic shifting in polymer-producing wastewater bacterium."],"submitter_affiliation":["northwestern university"],"submitter_name":["Xinyu Chen"],"organism_part":["Cell Pellet"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["Intracellular metabolite levels were determined from cell suspensions obtained during exponential growth phase. The suspensions were filtered and the cell-containing filters were immediately quenched by submerging them in a cold (4°C) 2-mL solution of methanol:acetonitrile:water (40:40:20). Solutions with the lysed cells were subsequently filter-centrifuged (Sigma-Aldrich Spin-X 0.22 μm filters). Aliquots of the supernatants were dried under nitrogen gas and re-suspended in LC-MS ultrapure water (Fisher Scientific, Pittsburgh, Pennsylvania) before analysis via LC-MS. Metabolite levels were normalized to biomass quantity at the time of sampling.
"],"organism":["Comamonas testosteroni KF-1"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS12449"],"author":["Ludmilla Aristilde. Northwestern university. 2145 Sheridan Road, Tech A316, Evanston, IL 60208-3109. ludmilla.aristilde@northwestern.edu. 847-491-2999."],"data_transformation_protocol":["All metabolite identification and isotopic enrichment were determined using the Metabolomics Analysis and Visualization Engine (MAVEN) software package (Clasquin et al., 2012). Corrections for natural abundance of 13C were conducted on the 13C-labeled fractions.
"],"study_factor":["Nitrogen level"],"submitter_email":["xinyuchen2024@u.northwestern.edu"],"sample_collection_protocol":["For glycogen quantification, both extracellular and intracellular samples were obtained at 6 to 8 time points throughout biomass growth. Sample preparation and enzymatic hydrolysis of glycogen were done with a commercially available glycogen assay kit (ab65620, Abcam, UK). Glucose in the samples was then quantified with LC-HRMS following a published protocol. For PHA quantification, we developed a LC-HRMS method to determine and quantify the following composition of PHA in C testosteroni following PHA hydrolysis: 3-hydroxybutyric acid (C4), 3-hydroxyvaleric acid (C5), 3-hydroxyhexanoic acid (C6), 3-hydroxyoctanoic acid (C8), 3-hydroxydecanoic acid (C10), 3-hydroxydodecanoic acid (C12), 3-hydroxytetradecanoic acid (C14) and 3-hydroxyhexadecanoic acid (C16) (Extended Data Fig. 3 and Supplementary Table 5). For this analysis, 8 mg of lyophilized cells were collected at different time points (in three biological replicates) throughout biomass growth under the different nutrient conditions. The cells were suspended in 1 mL of 2 M NaOH, sealed, and heated at 100 °C for 5 hours using a heating block. After cooling to room temperature, 1 mL of 2 M HCl was added to neutralize the solution. The mixture was then filtered through a 0.45-µm polyethersulfone membranes filters (Millipore; Burlington, MA), diluted 100- or 200-fold using water (LC/MS Grade, Fisher Scientific, USA), and subjected to LC-HRMS analysis. PHA monomer separation and analysis followed a previously described LC-MS-based metabolomics protocol, with minor modifications. The flow rate was 180 µL min-1 during the entire 32 min sample run. The solvent gradient with the respect to solvent A was the following: 0 min, 100%; 2.5 min, 100%; 5 min, 80%; 7.5 min, 80%; 10 min, 45%; 12 min, 45%; 15 min, 5%; 20 min, 5%; 23 min, 5%; 26 min, 0%; 32 min, 0% was run. All chemicals for PHA standards were purchased from Sigma Aldrich.
"],"omics_type":["Metabolomics"],"study_design":["ultra-performance liquid chromatography-mass spectrometry","Comamonas testosteroni KF-1","Nutrient","poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate)"],"curator_keywords":["ultra-performance liquid chromatography-mass spectrometry","Comamonas testosteroni KF-1","Nutrient","poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate)"],"mass_spectrometry_protocol":["An electrospray ionization interface was used to direct column eluent to the mass spectrometer. Because the ion pairing agent tributylamine will cause ion suppression in positive mode, the instrument was operated in negative mode only. Initial instrument optimization (tuning) was done by infusing a mixture of malate (m/z 133.0142), ATP (m/z 505.9885), and coenzyme A (m/z 766.1079), each at 1 μg/mL at a flow rate of 200 μg/mL, using a 11Plus syringe pump (Harvard Apparatus, Boston, MA). Various instrumental settings were optimized to maximize the signal with the final parameters as follows: sheath gas flow rate 25 (arbitrary units), auxiliary gas flow rate 8 (arbitrary units), sweep gas flow rate 3 (arbitrary units), spray voltage 3 kV, capillary temperature 325 °C, capillary voltage −50 V, tube lens voltage −100 V. The instrument was mass calibrated using the polytyrosine-1,3,6 standards every 3 days. Anal. Chem. 2010, 82, 8, 3212–3221
"],"additional_accession":[]},"is_claimable":false,"name":"Nutrient depletion and systems-wide metabolic shifting in polymer-producing wastewater bacterium","description":"Inorganic nutrient deficiency stimulates polymer synthesis in bacterial producers relevant to wastewater treatment and biotechnology, but the metabolic mechanisms are poorly understood. Here, using a quantitative multi-omics analysis of the wastewater isolate Comamonas testosteroni during nitrogen and phosphorus depletion, we uncover widespread metabolic rebalancing of carbon and energy fluxes beyond the synthesis of polyhydroxyalkanoate and glycogen, thereby revealing other key metabolic phenotypes important for surviving nutrient limitation with additional relevance to biotechnology applications.
","dates":{"publication":"2026-09-09","submission":"2025-05-02"},"accession":"MTBLS12449","cross_references":{}}