Mass spectrometry datasets on TFEB proteostasis
Data files
Apr 21, 2025 version files 1.68 GB
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MSB-6066_UPMC_Lear_082117_TFEB-GFP_PD_for_E3.xlsx
121.44 KB
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MSB-6185_UPMC_Lear_092917__Phospho-proteomics.xlsx
33.21 KB
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MSB29110IPTFEBBand_08.mzML
76.19 MB
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MSB29110IPTFEBBand_08.raw
200.35 MB
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MSB29111IPEBand_10.mzML
74.87 MB
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MSB29111IPEBand_10.raw
198.83 MB
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MSB29792A.mzML
615.71 MB
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MSB29792A.raw
218.01 MB
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MSB29793A.mzML
85.96 MB
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MSB29793A.raw
205.94 MB
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README.md
4.05 KB
Abstract
TFEB-interacting proteins were determined through immunoprecipitation of TFEB from MLE-12 cells. We compared proteins identified on the TFEB-EGFP resin to the control resin to find candidate binding partners of TFEB. These experiments identified DCAF7 as a binding partner of TFEB. Phosphoproteomics analysis revealed phosphorylation sites in TFEB protein.
Dataset DOI: 10.5061/dryad.p2ngf1w2s
Description of the data and file structure
These datasets describe experiments to find binding partners and phosphorylation sites in TFEB. Binding partners were determined using Immunoprecipitation (IP) followed by mass spectrometry in MLE-12 cells. Phosphorylation sites in TFEB were determined by gel purification of TFEB from lysate derived from MLE-12 cells that were treated with carfilzomib (4 hour treatment at 1µM), or vehicle control.
Files and variables
File: MSB29110IPTFEBBand_08.mzML
Description: Immunoprecipitation of TFEB-GFP, processed mass spectrum provided in mzML format.
File: MSB29111IPEBand_10.mzML
Description: Immunoprecipitation of Empty-Vector GFP (Control), processed mass spectrum provided in mzML format.
File: MSB29110IPTFEBBand_08.raw
Description: Immunoprecipitation of TFEB-GFP, raw data (Thermo format).
File: MSB29111IPEBand_10.raw
Description: Immunoprecipitation of Empty-Vector GFP (Control), raw data (Thermo format).
File: MSB29792A.mzML
Description: Mass spectroscopy of TFEB protein excised from polyacrylamide gel electrophoresis band. This sample was derived from MLE-12 cells treated with carfilzomib. This is processed mass spectrum data provided in mzML format.
File: MSB29792A.raw
Description: Mass spectroscopy of TFEB protein excised from polyacrylamide gel electrophoresis band. This sample was derived from MLE-12 cells treated with carfilzomib. This is raw data (Thermo format).
File: MSB29793A.mzML
Description: Mass spectroscopy of TFEB protein excised from polyacrylamide gel electrophoresis band. This sample was derived from control MLE-12 cells. This is processed mass spectrum data provided in mzML format.
File: MSB29793A.raw
Description: Mass spectroscopy of TFEB protein excised from polyacrylamide gel electrophoresis band. This sample was derived from control MLE-12 cells. This is raw data (Thermo format).format.
File: MSB-6185_UPMC_Lear_092917__Phospho-proteomics.xlsx
Description: Results of Mascot search (Matrix Science) for the Phospho-proteomics experiment.
Variables
- Identified Protein (Description of the identified protein).
- Accession Number (Uniprot Accession information).
- Molecular Weight of the identified protein.
- SPC 29792 Total number of Matching spectra for the given Protein (CFZ treatment)
- SPC 29793 Total number of Matching spectra for the given Protein (Control treatment)
File: MSB-6066_UPMC_Lear_082117_TFEB-GFP_PD_for_E3.xlsx
Description: Results of Mascot search (Matrix Science) for the E3 pulldown experiment.
Two sheets are provided, each with the same column definitions:
Protein Report Sheet: A list of all proteins identified in the Mascot search.
Filtered Protein Report Sheet: A list of all proteins from the Protein Report Sheet that have at least 5 matching spectra (SpC) in the IP-TFEB experiment, and was either not detected in the IP-Ctrl experiment or at least 4-fold higher in the IP-TFEB experiment relative to the IP-Ctrl experiment.
Variables
- Identified Proteins (Number of Identified Proteins): A Description of the identified proteins.
- Accession Number: Uniprot Accession information.
- Molecular Weight: Molecular Weight of the identified protein.
- SpC 29110 (IP-TFEB): Total number of Matching spectra for the given Protein in the TFEB pulldown experiment.
- SpC 29111 (IP Ctrl): Total number of Matching spectra for the given Protein in the empty vector pulldown experiment.
Code/software
These mass spectra can be searched to identify proteins of interest using search algorithms such as ProLucid (https://github.com/bathyg/Prolucid_gui) or MaxQuant/Andromeda (https://maxquant.org).
Thermo Raw spectra files were converted to mzML format using ProteoWizard/MSConvertGUI.exe, version 3.0.25084-03e34a9.
Immunoprecipitation of TFEB
TFEB-EGFP expressing cells ore Empty vector control cells were precipitated with GFP-Trap magnetic resin (ChromoTek). Resin was washed and sent for mass spectrometry analysis. Samples were processed and analyzed by MSBioworks (Ann Arbor, MI). Briefly, samples were boiled at 100°C for 15 minutes in 60 μL of 1.5X LDS buffer. The beads were removed and half of each submitted sample was processed by SDS-PAGE using a 10% Bis-Tris NuPAGE gel (Invitrogen) with the MES buffer system. The mobility region was excised into 10 equal sized segments and in-gel digestion was performed on each using a robot (ProGest, DigiLab) with the following protocol: washed with 25 mM ammonium bicarbonate followed by acetonitrile, reduced with 10 mM dithiothreitol at 60°C followed by alkylation with 50 mM iodoacetamide at RT, digested with sequencing grade trypsin (Promega) at 37°C for 4 h, quenched with formic acid and the supernatant was analyzed directly without further processing. Half of each digested sample was analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Data were filtered using 1% protein and peptide FDR and requiring at least two unique peptides per protein.
Phosphoproteomics
TFEB-EGFP MLE-12 cells were treated with vehicle or carfilzomib (1 μM for 4 h) prior to trypsinization and snap freezing. Cell lysate was sent to MSBioworks. Briefly, Samples were boiled at 100°C for 15 minutes in 60 μL of 1.5X LDS buffer. Beads were removed and half of each submitted sample was processed by SDS-PAGE using a 4-12% Bis-Tris NuPAGE gel (Invitrogen) with the MOPS buffer system. The target band (TFEB+GFP) was excised and processed by in-gel digestion using a robot (ProGest, DigiLab) Data were filtered using 1% protein and peptide FDR and requiring at least two unique peptides per protein.