DOI: 10.5061/dryad.p2ngf1w2s
title: Mass spectrometry datasets on TFEB proteostasis

description: 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.
description: 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.
description: # Mass spectrometry datasets on TFEB proteostasis. Dataset DOI:
 [10.5061/dryad.p2ngf1w2s](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.