Summary of Study ST002007

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR001272. The data can be accessed directly via it's Project DOI: 10.21228/M89402 This work is supported by NIH grant, U2C- DK119886.


This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002007
Study TitleIsotope tracing analysis of stress erythroid progenitors
Study SummaryIsotope tracing analysis to study the intracellular metabolic changes of progenitors during the expansion stage of stress erythropoiesis and assess the effect of 1400w treatment.
Pennsylvania State University
DepartmentVeterinary and Biomedical Sciences
LaboratoryPaulson Lab
Last NameRuan
First NameBaiye
Address228 AVBS Building Shortlidge Road University Park, PA 16802
Submit Date2021-12-02
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2022-12-12
Release Version1
Baiye Ruan Baiye Ruan application/zip

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Project ID:PR001272
Project DOI:doi: 10.21228/M89402
Project Title:Metabolic profiles of murine stress erythroid progenitors
Project Summary:Inflammation alters hematopoiesis, skewing production to generate myeloid effector cells at the expense of steady state erythropoiesis. To compensate, stress erythropoiesis is induced to maintain homeostasis until the inflammation is resolved. Unlike the constant production of steady state erythropoiesis, stress erythropoiesis generates a bolus of new erythrocytes by first producing immature progenitor cells, which then transition to committed erythroid progenitors and differentiate. We hypothesize that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism. Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation. In contrast, the transition to differentiation is marked by decreased Nos2 expression and a change in metabolism to support induction of the erythroid gene expression program. These data support a model where increased pro-inflammatory signals inhibit steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis.
Institute:Pennsylvania State University
Department:Veterinary and Biomedical Sciences
Laboratory:Paulson Lab
Last Name:Ruan
First Name:Baiye
Address:228 AVBS Building, Shortlidge Road, University Park, PA 16802


Subject ID:SU002088
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090


Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Mouse_strain Culture_stage Time_point(days) Treatment
SA187821EM1-2wild type C57BL/6 Expansion stage 1 untreated
SA187822EM1-1wild type C57BL/6 Expansion stage 1 untreated
SA187823EM1-5wild type C57BL/6 Expansion stage 1 untreated
SA187824EM1-3wild type C57BL/6 Expansion stage 1 untreated
SA187825EM1-4wild type C57BL/6 Expansion stage 1 untreated
SA187826EM3-5wild type C57BL/6 Expansion stage 3 untreated
SA187827EM3-4wild type C57BL/6 Expansion stage 3 untreated
SA187828EM3-2wild type C57BL/6 Expansion stage 3 untreated
SA187829EM3-3wild type C57BL/6 Expansion stage 3 untreated
SA187830EM3-1wild type C57BL/6 Expansion stage 3 untreated
SA1878311400w-3wild type C57BL/6 Expansion stage 5 1400w
SA1878321400w-1wild type C57BL/6 Expansion stage 5 1400w
SA1878331400w-2wild type C57BL/6 Expansion stage 5 1400w
SA1878341400w-4wild type C57BL/6 Expansion stage 5 1400w
SA1878351400w-5wild type C57BL/6 Expansion stage 5 1400w
SA187841EM5-3wild type C57BL/6 Expansion stage 5 untreated
SA187842EM5-1wild type C57BL/6 Expansion stage 5 untreated
SA187843EM5-2wild type C57BL/6 Expansion stage 5 untreated
SA187844EM5-4wild type C57BL/6 Expansion stage 5 untreated
SA187845EM5-5wild type C57BL/6 Expansion stage 5 untreated
SA187836Veh-4wild type C57BL/6 Expansion stage 5 Vehicle (DMSO)
SA187837Veh-5wild type C57BL/6 Expansion stage 5 Vehicle (DMSO)
SA187838Veh-3wild type C57BL/6 Expansion stage 5 Vehicle (DMSO)
SA187839Veh-1wild type C57BL/6 Expansion stage 5 Vehicle (DMSO)
SA187840Veh-2wild type C57BL/6 Expansion stage 5 Vehicle (DMSO)
Showing results 1 to 25 of 25


Collection ID:CO002081
Collection Summary:Unfractionated wild-type bone marrow cells were cultured in regular stress erythropoiesis expansion medium (SEEM). At indicated time point, stress erythroid progenitor cells were harvested, washed with PBS, and re-cultured in SEEM supplemented with 25 mM uniformly labeled [13C]-Glucose (Sigma-Aldrich) for 24 hrs. Cells were counted, washed with PBS and snap frozen in liquid nitrogen.
Sample Type:Stem cells


Treatment ID:TR002100
Treatment Summary:To assess the role of iNOS in progenitor metabolism, SEEM cultures were treated with 10 μM 1400w or vehicle (DMSO) at day 3 for 48 hrs, and then cells were re-cultured in SEEM with U-[13C]-Glucose for 24 hrs.
Treatment Compound:1400w
Treatment Dose:10 μM
Treatment Vehicle:DMSO
Cell Media:SEEM

Sample Preparation:

Sampleprep ID:SP002094
Sampleprep Summary:Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Combined analysis:

Analysis ID AN003270
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Dionex Ultimate 3000
Column Waters XSelect HSS C18 (250 x 4.6mm)
MS instrument type Orbitrap
MS instrument name Thermo Exactive Plus Orbitrap
Units peak area


Chromatography ID:CH002414
Chromatography Summary:The sample run order was randomized to reduce bias from instrument drift. 10 µl sample was subjected to LC-MS analysis on a Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Reversed-phase chromatography mode was used to separate compounds on a Xselect C18 HSS column (Waters) with solvent A (97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid ) and solvent B (methanol). The flow rate was 200 µl/min, and the total run time was 25 min. The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. The mass spectrometer was operated in a negative-ion mode at a resolution of 140,000 at m/z 200 and with a scan range of 85 to 1000 m/z.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters XSelect HSS C18 (250 x 4.6mm)
Flow Gradient:The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B.
Flow Rate:200 ul/min
Solvent A:97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine
Solvent B:100% methanol
Chromatography Type:Reversed phase


MS ID:MS003042
Analysis ID:AN003270
Instrument Name:Thermo Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Comments:Raw data files were converted to .mzML file format using the ProteoWizard software, and they were analyzed by the MS-DIAL software. Metabolites were identified by comparison to an in-house reference library of pure metabolite standards which included mass-to-charge ratio (m/z) and retention time. For quantification of metabolite abundance, peak areas of identified metabolites were first normalized to the internal standard chlorpropamide, and then normalized to cell numbers from each sample. Data were analyzed using R and Cytoscape software.