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
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.
| Study ID | ST001438 |
| Study Title | Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited samples - MSC |
| Study 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. 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 Name | Fernandez |
| First Name | Facundo |
| Address | 901 Atlantic Dr NE, Atlanta, GA, 30332, USA |
| fernandez@gatech.edu | |
| Phone | 404-385-4432 |
| Submit Date | 2020-08-02 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | MS(Dir. Inf.) |
| Release Date | 2020-09-14 |
| Release Version | 1 |
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 |
|---|---|---|
| SA122068 | Blank_3 | Blank |
| SA122069 | Blank_2 | Blank |
| SA122070 | QC_C1 | QC |
| SA122071 | QC_C3 | QC |
| SA122072 | QC_B3 | QC |
| SA122073 | QC_B2 | QC |
| SA122074 | QC_A1 | QC |
| SA122075 | QC_A2 | QC |
| SA122076 | S6_2 | Stimulated |
| SA122077 | S1_1 | Stimulated |
| SA122078 | S6_1 | Stimulated |
| SA122079 | S3_1 | Stimulated |
| SA122080 | S2_3 | Stimulated |
| SA122081 | S2_1 | Stimulated |
| SA122082 | S1_2 | Stimulated |
| SA122083 | S3_2 | Stimulated |
| SA122084 | S4_2 | Stimulated |
| SA122085 | S5_3 | Stimulated |
| SA122086 | S5_1 | Stimulated |
| SA122087 | S4_3 | Stimulated |
| SA122088 | U6_3 | Unstimulated |
| SA122089 | U3_1 | Unstimulated |
| SA122090 | U2_3 | Unstimulated |
| SA122091 | U2_2 | Unstimulated |
| SA122092 | U1_2 | Unstimulated |
| SA122093 | U1_1 | Unstimulated |
| SA122094 | U3_3 | Unstimulated |
| SA122095 | U4_1 | Unstimulated |
| SA122096 | U5_3 | Unstimulated |
| SA122097 | U5_2 | Unstimulated |
| SA122098 | U4_2 | Unstimulated |
| SA122099 | U6_1 | Unstimulated |
| 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 | ESI |
| 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: | ESI |
| MS Comments: | See attached Method file |
| Ion Mode: | POSITIVE |
