MassIVE MSV000084214

Partial Public

SILAC diGlycine proteomics in C. elegans to identify ULP-3 targets upon DNA damage.

Subscribe Comment Add Reanalysis

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. [doi:10.25345/C5NS9R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: nedd8, DNA damage, apoptosis, C. elegans

Contact

Principal Investigators:
(in alphabetical order) 		Dimitris Xirodimas, Cell Biology Research Centre of Montpellier, France
Submitting User: dxirodimas

Publications

Bailly AP, Perrin A, Serrano-Macia M, Maghames C, Leidecker O, Trauchessec H, Martinez-Chantar ML, Gartner A, Xirodimas DP.
The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity.
Cell Rep. 2019 Oct 1;29(1):212-224.e8.

Number of Files:
Total Size:
Spectra:
Subscribers:
 
Owner Reanalyses
Experimental Design
    Conditions:
    Biological Replicates:
    Technical Replicates:
 
Identification Results
    Proteins (Human, Remapped):
    Proteins (Reported):
    Peptides:
    Variant Peptides:
    PSMs:
 
Quantification Results
    Differential Proteins:
    Quantified Proteins:
 
Browse Dataset Files
 
FTP Download Link (click to copy):

Species

Instrument

Modifications

- Dataset Reanalyses

+ Dataset History

Click here to queue conversion of this dataset's submitted spectrum files to open formats (e.g. mzML). This process may take some time.

When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.