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Ubiquitin remnant motif profiling (Ubiquitomics) in VACV infected HeLa cells

ABSTRACT: HeLa cells were pretreated with MG132, inoculated with VACV-Cop at an MOI of 3 for 1 h, and posttreated with MG132 again. Cells were lysed and extracted proteins were digested to peptides using trypsin, and desalted peptides were subjected to automated diGly enrichment using the PTMScan ubiquitin remnant motif kit (Cell signaling) according to manufactuer's instructions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Vaccinia Virus Copenhagen (ncbitaxon:10249) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Olivier Julien Robert Ingham

PROVIDER: MSV000094020 | MassIVE | Mon Feb 05 17:31:00 GMT 2024

REPOSITORIES: MassIVE

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Project description: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

Project description:We found a conserved role for the deNEDDylating enzyme NEDP1 (ULP-3 in C. elegans) in the DNA damage induced apoptosis. To gain mechanistic insights into the role of ULP-3 in the IR-induced apoptosis, we devised an unbiased proteomics approach to discover potential NEDD8 targets for ULP-3 upon DNA damage. Identification of diglycine (diGly) remnants left on lysine residues upon trypsin digestion of proteins by mass spectrometry demonstrates their modification with ubiquitin, NEDD8 or ISG15 (Kessler, 2013; Ordureau et al., 2015). We hypothesised that changes in the diGly signature upon deletion of the de-NEDDylating enzyme ULP-3, should specifically indicate changes in the NEDD8 modification repertoire. We combined Stable Isotope Labelling with Amino acid in nematodes (SILAC) (Larance et al., 2011) with the use of antibodies that recognise the diGly remnant on modified peptides (Kessler, 2013; Ordureau et al., 2015; Xu et al., 2010). Wild type and ulp-3 deleted worms were labelled with Light (K0R0) and Heavy (K8R10) isotopes respectively. To specifically address the effect of ULP-3 on the NEDD8 proteome upon DNA damage, both sets of worms were exposed to IR. Synchronised worms were grown on nitrogen free nematode growth medium (NGM-N) as described previously (Larance et al., 2011) seeded with SLE1 bacteria labelled with Light (K0R0) and Heavy (K8R10) Isotopes (Eurisotope). Larvae stage 1 synchronised worms were grown until late adult stage and synchronised again by bleaching before growing them on 9cm NGM-N plates seeded with SILAC bacteria (SLE1). Approximately 10 plates of 4000 worms each were used to grow synchronised and labelled worms until late L4-young adult stage that were irradiated with 90Gy of IR using a Blood-Xrad apparatus. Labelled worms proteins were extracted in 50mM Hepes-NaOH pH8.0, Urea 9M using a bead beater and zirconium 0.7mm beads. Extracts were mixed in 1:1 ratio before immunoprecipitation with the anti-diGly antibody followed by MS/MS, performed by Cell Signaling Technology.

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Ubiquitin remnant motif profiling (Ubiquitomics) in VACV infected HeLa cells

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