Summary of Study ST003666
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 PR002274. The data can be accessed directly via it's Project DOI: 10.21228/M8PN92 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 | ST003666 |
| Study Title | TGFB1-mediated intercellular signaling fuels cooperative cellular invasion |
| Study Summary | Conditioned media from SaGA-derived leader and follower subpopulations (Khatib 2023, Konen 2017) were collected and analyzed using mass spectrometry-based proteomics to assess differences in cell secretion profiles. Label-free quantification was employed to compare spectra against the 2020 human UniProtKB/Swiss-Prot database (20,379 target sequences). This analysis identified 1,174 proteins. Our findings revealed that the follower subpopulation produces and secretes TGFB1 and TGFB-related factors. Notably, the leader subpopulation secretes FN1 and ALCAM, which are downstream effectors of TGFB-mediated signaling. |
| Institute | Emory University |
| Last Name | Khatib |
| First Name | Tala |
| Address | 201 Dowman Dr, Atlanta, GA 30322 |
| tkhatib@emory.edu | |
| Phone | 9102622080 |
| Submit Date | 2024-11-07 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-01-20 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002274 |
| Project DOI: | doi: 10.21228/M8PN92 |
| Project Title: | TGFB1-mediated intercellular signaling fuels cooperative cellular invasion |
| Project Type: | Proteomics Mass Spectrometry on conditioned media |
| Project Summary: | SaGA-derived (Khatib 2023, Konen 2017) leader and follower subpopulation conditioned media was collected and sequenced via Mass spectrometry/proteomics to compare differences in cell secretion from subpopulations originally isolated from the same parental population. |
| Institute: | Emory University |
| Department: | Hematology and Oncology |
| Laboratory: | Marcus Lab |
| Last Name: | Khatib |
| First Name: | Tala |
| Address: | 201 Dowman Dr, Atlanta, GA 30322 |
| Email: | tkhatib@emory.edu |
| Phone: | 9102622080 |
| Funding Source: | This work was supported by NIH NCI grants R01CA250422 (A.I.M.), R01CA247367 (A.I.M.), R01CA236369 (A.I.M.), PO1CA257906 (A.I.M.), R50CA265345 (J.K.M.), National Science Foundation Graduate Research Fellowship (NSF-GRFP 1937971), and an Imagine, Innovate, Impact (I3) Research award from the Emory School of Medicine through the Georgia CTSA NIH award UL1-TR002378 (A.I.M. and H.G.). |
| Contributors: | Janna Mouw, Tala Khatib |
Subject:
| Subject ID: | SU003798 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | H1299 cell line |
| Age Or Age Range: | 45 |
| Gender: | Male |
| Cell Strain Details: | H1299 cell line |
| Cell Passage Number: | 3,4,5 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Type |
|---|---|---|
| SA401291 | Follower_1 | Conditioned media |
| SA401292 | Follower_2 | Conditioned media |
| SA401293 | Follower_3 | Conditioned media |
| SA401294 | Follower_4 | Conditioned media |
| SA401295 | Leader_1 | Conditioned media |
| SA401296 | Leader_2 | Conditioned media |
| SA401297 | Leader_3 | Conditioned media |
| SA401298 | Leader_4 | Conditioned media |
| Showing results 1 to 8 of 8 |
Collection:
| Collection ID: | CO003791 |
| Collection Summary: | Cells were initially cultured in standard media for 24 h and then replaced with defined media after two rinses with 1X PBS. After 48 h, the media was collected and centrifuged at 250 g for 5 min to remove any detached cells, followed by a 20 min centrifugation at 2000 g to eliminate apoptotic bodies. The media was then concentrated using the Amicon ultra-15 centrifugal filter unit with a 3-kDA cut off and centrifugation at 4000 g at 4℃ for a final volume of 250 μL. Samples were flash-frozen before storage at -80℃. |
| Sample Type: | Lung |
Treatment:
| Treatment ID: | TR003807 |
| Treatment Summary: | LC-MS/MS was performed on the secreted media content of adherent leader and follower cell populations in 2D culture. Cells were initially cultured in standard media for 24 h and then replaced with defined media [1X insulin-transferrin-selenium-ethanolamine, 0.5 mg/mL hydrocortisone, 1 ng/mL cholera toxin, 50 nM O-phosphoryl-ethanolamine, and 5 nM triiodothyronine] after two rinses with 1X PBS. After 48 h, the media was collected and concentrated for LC-MS/MS. |
| Treatment Protocol Filename: | Protocol_tkhatib.pdf |
Sample Preparation:
| Sampleprep ID: | SP003805 |
| Sampleprep Summary: | Protein normalization, digestion, and LC-MS/MS data acquisition and analysis were conducted at the Emory Integrated Proteomics Core. |
| Sampleprep Protocol Filename: | Protocol_tkhatib.pdf |
Combined analysis:
| Analysis ID | AN006023 |
|---|---|
| Chromatography ID | CH004576 |
| MS ID | MS005734 |
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters NanoAcquity |
| Column | Waters ACQUITY UPLC CSH C18 (150 x 2.1 mm,1.7 μm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Orbitrap Fusion Lumos Tribrid |
| Ion Mode | POSITIVE |
| Units | LQF Intensity |
Chromatography:
| Chromatography ID: | CH004576 |
| Instrument Name: | Waters NanoAcquity |
| Column Name: | Waters ACQUITY UPLC CSH C18 (150 x 2.1 mm,1.7 μm) |
| Column Temperature: | 60℃ |
| Flow Gradient: | 60 min gradient |
| Flow Rate: | 1.25 μL/min |
| Solvent A: | 99.9% water; 0.1% formic acid |
| Solvent B: | 99.9% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005734 |
| Analysis ID: | AN006023 |
| Instrument Name: | Thermo Orbitrap Fusion Lumos Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The data acquisition by LC-MS/MS protocol was adapted from a published procedure (Seyfried, Dammer et al. 2017) and was performed by the Integrated Proteomics Core Facility at Emory University. Derived peptides were resuspended in loading buffer (0.1% trifluoroacetic acid). Peptide mixtures were separated on a self-packed 1.7 µm CSH waters resin column (15 cm x 100 µm internal diameter (ID); New Objective, Woburn, MA) attached to a Dionex 3000 RSLCnano system and were monitored on a Orbitrap Fusion Lumos Mass Spectrometer (ThermoFisher Scientific, San Jose, CA). Elution was performed over a 60 min gradient at a rate of 1.25 µL/min (buffer A: 0.1% formic acid in water, buffer B: 0.1 % formic acid in acetonitrile): The gradient started with 1% buffer B and went to 5% in 6 sec followed by 30% in 53 minutes, and then increased to 99% within 1 minute and staying at 99% for another 4 minutes before ending at 1% for 2 minutes. The mass spectrometer was operated in data dependent mode in top speed mode with a cycle time of 3 seconds. Survey scans were collected in the Orbitrap with a 120,000 resolution, 400 to 1600 m/z range, 400,000 automatic gain control (AGC), 246 ms max injection time and rf lens at 30%. Higher energy collision dissociation (HCD) tandem mass spectra were collected in the Orbitrap with a 15,000 resolution, collision energy of 35%, an isolation width of 1.6 m/z, AGC target of 100000, and a max injection time of 54 ms. Dynamic exclusion was set to 30 seconds with a 10 ppm mass tolerance window. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | LC_MS_MS_protocol.pdf |
