Dataset Information

Mouse Cerebellum DNA-Damage 2D LC-MS/MS

ABSTRACT: Whole mice cerebella tissue was lysed, reduced, alkylated and digested. Three time points of control and KO tissue was labeled using dimethyl isotope labeling. After combination SCX fractionation was performed followed by RP LC-MS/MS analysis using an Orbitrap Velos and top 10 HCD fragmentation.

REANALYSED by: PAe005208

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Erik L. de Graaf

PROVIDER: PXD000046 | Pride | 2014-03-05

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	ERCC1_F1_16w_F272509.dat	Other
	ERCC1_F1_26w_F272512.dat	Other
	ERCC1_F1_8w_F272505.dat	Other
	ERCC1_F2_16w_F272517.dat	Other
	ERCC1_F2_26w_F272519.dat	Other

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Publications

Spatio-temporal analysis of molecular determinants of neuronal degeneration in the aging mouse cerebellum.

de Graaf Erik L EL Vermeij Wilbert P WP de Waard Monique C MC Rijksen Yvonne Y van der Pluijm Ingrid I Hoogenraad Casper C CC Hoeijmakers Jan H J JH Altelaar A F Maarten AF Heck Albert J R AJ

Molecular & cellular proteomics : MCP 20130211 5

The accumulation of cellular damage, including DNA damage, is hypothesized to contribute to aging-related neurodegenerative changes. DNA excision repair cross-complementing group 1 (Ercc1) knock-out mice represent an accepted model of neuronal aging, showing gradual neurodegenerative changes, including loss of synaptic contacts and cell body shrinkage. Here, we used the Purkinje cell-specific Ercc1 DNA-repair knock-out mouse model to study aging in the mouse cerebellum. We performed an in-depth ...[more]

PMID: 23399551

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Project description:Cerebella from wild-type and cGKI knockout mice, on 129/Sv background, were subjected to protein digestion using trypsin o/n and chemically labeled with dimethyl labeling. After labeling the samples were mixed and subjected to both strong cation exchange and phosphopeptide enrichment. The SCX fractions and the enriched phosphopeptides were then analyzed on an Orbitrap mass spectrometer. Data analysis: For each raw data file recorded by the mass spectrometer, peak lists were generated using Proteome Discoverer (version 1.3, Thermo Scientific, Bremen, Germany) using a standardized workflow. Peak lists, generated in Proteome Discoverer, were searched against a Swiss-Prot database (version 2.3.02, taxonomy Mus musculus, 32402 protein entries) supplemented with frequently observed contaminants, using Mascot (version 2.3.02 Matrix Science, London, UK). The database search was performed by using the following paramenters: a mass tolerance of 50 ppm for the precursor masses and +/-0.6 Da for CID/ETD fragment ions and +/-0.05 Da for HCD fragments. Enzyme speciﬁcity was set to Trypsin with 2 missed cleavages allowed. Carbarmidomethylation of cysteines was set as fixed modification, oxidation of methionine and dimethyl labeling (L, I, H) of lysine residues and N termini, and phosphorylation (S, T, Y) (for the phosphoproteome analysis) were used as variable modifications. Percolator was used to ﬁlter the PSMs for <1% false discovery-rate. Phosphorylation sites were localized by applying phosphoRS (pRS) (v2.0). Triplex dimethyl labeling was used as quantification method, with a mass precision for the 2 ppm for consecutive precursor mass scan. A retention time tolerance of 0.5 min was used to account effect of deuterium on retention time. To further filter for high quality data we used the following parameters: high confidence peptide spectrum matches, minimal Mascot score of 20, minimal peptide length of 6 and only unique rank 1 peptide. For the phosphopeptides analysis, the search rank 1 peptide was added to the previous filters. For the identification and quantitation of the proteins, only the unique peptides were considered.