Summary of study ST001438

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000988. The data can be accessed directly via it's Project DOI: 10.21228/M80680 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study IDST001438
Study TitleSub-nanoliter metabolomics via mass spectrometry to characterize volume-limited samples - MSC
Study SummaryThe human metabolome provides a window into the mechanisms and biomarkers of various diseases. However, because of limited availability, many sample types are still difficult to study by metabolomic analyses. Here, we present a new mass spectrometry (MS)-based metabolomics strategy that only consumes sub-nanoliter sample volumes. The approach consists of combining a customized metabolomics workflow with a pulsed MS ion generation method, known as triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi nanoESI) MS. A second example to illustrate TENGi MS capabilities involves rare cell metabolomics of cultured mesenchymal stromal cells (MSCs), a cell type that has shown potential for treating a variety of chronic diseases. Examination of metabolic changes of MSCs cultured under conditions that may impact in vitro therapeutic activity, such as aggregate culture, or preconditioning with interferon gamma (IFN- γ)13, is critical for identifying attributes of cell quality. Reducing cell numbers required to perform MSC metabolomic analysis is essential for improving the manufacturing of highly therapeutic MSCs without significantly impeding production.
Institute
Georgia Institute of Technology
Last NameFernandez
First NameFacundo
Address901 Atlantic Dr NE, Atlanta, GA, 30332, USA
Emailfernandez@gatech.edu
Phone404-385-4432
Submit Date2020-08-02
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailESI
Release Date2020-09-14
Release Version1
Facundo Fernandez Facundo Fernandez
https://dx.doi.org/10.21228/M80680
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR000988
Project DOI:doi: 10.21228/M80680
Project Title:Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited samples
Project Summary:The human metabolome provides a window into the mechanisms and biomarkers of various diseases. However, because of limited availability, many sample types are still difficult to study by metabolomic analyses. Here, we present a new mass spectrometry (MS)-based metabolomics strategy that only consumes sub-nanoliter sample volumes. The approach consists of combining a customized metabolomics workflow with a pulsed MS ion generation method, known as triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi nanoESI) MS. Samples tested for this approach included exhaled breath condensates (EBC) collected from cystic fibrosis (CF) patients as well as in vitro-cultured human mesenchymal stromal cells (MSCs). Both test samples were only available in minimum amounts. Experiments showed that picoliter-volume spray pulses sufficed to generate high-quality spectral fingerprints, which increased the information density produced per unit sample volume. This TENGi nanoESI strategy has the potential to fill in the gap in metabolomics where liquid chromatography-MS-based analyses cannot be applied. Our method could open up new avenues for future investigations into understanding metabolic changes caused by diseases or external stimuli.
Institute:Georgia Institute of Technology
Last Name:Fernandez
First Name:Facundo
Address:901 Atlantic Dr NE, Atlanta, GA, 30332, USA
Email:fernandez@gatech.edu
Phone:404-385-4432

Subject:

Subject ID:SU001512
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Group
SA122068Blank_3Blank
SA122069Blank_2Blank
SA122070QC_C1QC
SA122071QC_C3QC
SA122072QC_B3QC
SA122073QC_B2QC
SA122074QC_A1QC
SA122075QC_A2QC
SA122076S6_2Stimulated
SA122077S1_1Stimulated
SA122078S6_1Stimulated
SA122079S3_1Stimulated
SA122080S2_3Stimulated
SA122081S2_1Stimulated
SA122082S1_2Stimulated
SA122083S3_2Stimulated
SA122084S4_2Stimulated
SA122085S5_3Stimulated
SA122086S5_1Stimulated
SA122087S4_3Stimulated
SA122088U6_3Unstimulated
SA122089U3_1Unstimulated
SA122090U2_3Unstimulated
SA122091U2_2Unstimulated
SA122092U1_2Unstimulated
SA122093U1_1Unstimulated
SA122094U3_3Unstimulated
SA122095U4_1Unstimulated
SA122096U5_3Unstimulated
SA122097U5_2Unstimulated
SA122098U4_2Unstimulated
SA122099U6_1Unstimulated
Showing results 1 to 32 of 32

Collection:

Collection ID:CO001507
Collection Summary:Bone marrow-derived MSCs (RoosterBio Inc., Lot #000139) were expanded for two passages in culture after being received. They were frozen in ~5x105 aliquots in Cryostor CS10 freeze media (BioLife). Frozen aliquots were revived and plated in tissue culture polystyrene flasks (Corning) for 3-4 days prior to seeding onto test surfaces. MSCs were cultured in low-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, lot E16063), and 1% antibiotic/antimycotic solution (Gibco). Once confluent, MSCs were washed with sterile-filtered phosphate-buffered saline (PBS, Thermo Fisher) and detached from flasks using TrypLE express (Thermo Fisher). Dissociated cells were counted using a hemacytometer and replated at 13,000 cells/cm2 in T-75 tissue culture flasks. After overnight incubation, MSCs then were exposed to 48 hours of culture media (control conditions), or culture media supplemented with 50 ng/mL IFN- γ (Thermo Fisher). MSCs were then washed with PBS and trypsinized as described above, and the number of harvested cells counted using a hemocytometer.
Sample Type:Mesenchymal stromal cells

Treatment:

Treatment ID:TR001527
Treatment Summary:MSCs were then washed with PBS and trypsinized as described above, and the number of harvested cells counted using a hemocytometer. MSCs were then resuspended in 155 mM ammonium acetate (Fluka) at a concentration of 1.6x106 cells/mL and aliquoted into 50-µL samples (8x104 cells per aliquot). Cells were then quenched by adding 200-µL MeOH into each sample vial and stored at -80 C until metabolite extraction. Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the supernatant, 200 µL was transferred into a new vial for lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final volume of 10 µL (for samples), and 18 µL (for QCs).

Sample Preparation:

Sampleprep ID:SP001520
Sampleprep Summary:Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the supernatant, 200 µL was transferred into a new vial for lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final volume of 10 µL (for samples), and 18 µL (for QCs).

Combined analysis:

Analysis ID AN002402
Analysis type MS
Chromatography type None (Direct infusion)
Chromatography system none
Column none
MS Type Other
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units Normalized Intensity (Intensity ratio against internal standard signal))

Chromatography:

Chromatography ID:CH001765
Instrument Name:none
Column Name:none
Chromatography Type:None (Direct infusion)

MS:

MS ID:MS002243
Analysis ID:AN002402
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:Other
MS Comments:See attached Method file
Ion Mode:POSITIVE
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