Mass spectrometry datasets on TFEB proteostasis

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.

Methods
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.