Dataset Information

The E3 ligase HECTD4 regulates COX-2 dependent tumor progression and metastasis

ABSTRACT: Dataset 1 Proteomics: This quantitative proteomics experiment aimed to characterize changes in global protein expression in order to identify HECTD4 substrates. For this, HECTD4-KD (n=3) and scrambled control MDA-MB-231 (n=3) cells were cultured in vitro under anchorage-independent conditions in ultra-low adherent plates for 3 days. The cells were treated with proteasome (MG132) and lysosome inhibitors (Bafilomycin-1, BafA1) for 6 hours and collected for proteomic analysis. Multiplexed mass spectrometry-based proteomics was performed using TMTpro barcoding reagents (PMID-33900084) and the SPS-MS3 method on an Orbitrap Lumos mass spectrometer. Prior to mass spectrometry analysis, the TMT set was fractionated offline by basic pH reversed-phase chromatography (bRPLC) into 24 fractions. Samples were labeled as follows 129c -Control_R1 130n - Control_R2 130c - Control_R3 131n - HECTD4 KD_R1 131c - HECTD4 KD_R2 132n - HECTD4 KD_R3 Dataset 2 DiGly Proteomics: This quantitative proteomics experiment aimed to characterize changes in protein ubiquitination in order to identify HECTD4 substrates. For this, HECTD4-KD (n=3) and scrambled control MDA-MB-231 (n=3) cells were cultured in vitro under anchorage-independent conditions in ultra-low adherent plates for 3 days. The cells were treated with proteasome (MG132) and lysosome inhibitors (Bafilomycine-1, BAF1) for 6 hours and collected for proteomic analysis. The Di-Gly peptides were enriched by co-immunoprecipitation of K-GG-modified peptides according to the manufacturers instructions (Cell Signaling PTMScan, Cat 59322). Multiplexed mass-spectrometry-based proteomics was performed using TMTpro barcoding reagents (PMID-33900084) using a high-resolution MS2 data-dependent acquisition method on an Orbitrap Lumos mass spectrometer. Before mass spectrometry analysis, the peptides were fractionated into seven fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific Cat 84868) according to the manufacturers instructions. The six fractions were each used once, and one was used twice for the mass spectrometry analysis. Samples were labeled as follows 129c -Control_R1 130n - Control_R2 130c - Control_R3 131n - HECTD4 KD_R1 131c - HECTD4 KD_R2 132n - HECTD4 KD_R3

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Wilhelm Haas Shyamala Maheswaran Daniel Haber