Proteome-wide analysis of USP14 substrates revealed its role in hepatosteatosis via stabilization of

Ubiquitinated peptide enrichment light labeled hela

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Ubiquitinated peptide enrichment . Light-labeled HeLa cell lysate and heavy- labeled USP14 KD HeLa cell lysate were mixed equally, digested and separated. Five percent of the peptides were used for protein quanti fi cation; whereas, the remaining peptides were used for ubiquitin peptides enrichment. Each fraction was dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and then incubated separately with pre-washed antibody beads (Cell Signaling Technology, Beverly, MA, USA and PTM Biolabs, Hangzhou, China) at 4 °C overnight with gently shaking. The bound peptides were eluted with 0.1% TFA and vacuum-dried followed by LC MS/MS analysis. Two biological replicates and two technical replicates were carried out for ubiquitinome analysis. Immunoprecipitation (IP) . Cells were lysed in lysis buffer for 20 min with gentle rocking at 4 °C. Lysates were cleared by centrifugation and then fi ltered through 0.45 μ m spin fi lters (Millipore) to further remove cell debris, and the resulting material was subjected to IP with 50 μ L anti-FLAG M2 af fi nity resin (Sigma) overnight at 4 °C. Resin-containing immune complexes was washed with ice-cold lysis buffer followed by Tris buffered saline (TBS) washes. Proteins were eluted with two-50 μ L 150 μ g/mL 3× Flag-peptide (Sigma) in TBS for 30 min, and the elution was pooled for a fi nal volume of 100 μ L. Proteins in each elution were precipitated with cold acetone. Three biological replicates were carried out. Nano-HPLC-MS/MS and mass spectrometry data analysis . The precipitated protein was digested with trypsin (trypsin: protein = 1:50 (w/w)) at 37 °C for 16 h. The peptides were then analyzed by an EASY-nLC 1000 system connected to an Q Exactive mass spectrometer and Orbitrap Fusion mass spectrometer (Thermo Fisher Scienti fi c, Waltham, MA, USA). The proteome data were acquired by Q Exactive with high resolution MS2 spectrum and the ubiquitinome and inter- actome data were acquired by Oribtrap Fusion with low resolution MS2 spectrum. Peptides were eluted from a homemade reverse-phase C18 column (75 μ m ID, 3 μ m particle size, Dikma Technologies Inc.) with a 70 min gradient of 7% to 80% buffer B (90% acetonitrile, 10% H 2 O, 0.1% formic acid) at a ow rate of 300 nL/ min. For the Q Exactive mass spectrometer, the automatic gain control (AGC) targets were 1.0e6 for full scan and 1.0e6 for MS/MS scan, respectively. Full MS spectra with an m/z range of 350 1300 were acquired with a resolution of 70,000 at m/z = 200 in pro fi le mode. Fragmentation of the 16 most intense precursor ions occurred at the high-energy collision dissociation (HCD) collision cell with a normalized collision energy of 28%, and tandem MS were obtained with a reso- lution of 17,500 at m/z = 200. Dynamic exclusion duration was 60 s and ions with a single charge or more than six charges were excluded from tandem mass frag- mentation. For the Orbitrap Fusion mass spectrometer, the automatic gain control (AGC) targets were 1.0e6 for full scan and 7e3 for MS/MS scan, respectively. Full MS spectra with an m/z range of 350
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  • Winter '19
  • Robert S Kiss
  • Fatty acid metabolism, Fatty acid synthase, FASN

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