Dataset Information

Label-free analysis of EAE vs Sham Spinal cord and PBMCs

ABSTRACT: Here we present the data obtained from a label-free quantitative proteomics analysis of soluble spinal cord extract derived from a mouse model of multiple sclerosis (EAE) and sham-induced mice. Samples were prepared offline using the FASP approach and then submitted for nano-LC-MS/MS analysis on an Orbitrap Velos instrument. After statistical evaluation of the data, 431 differentially expressed proteins (KS-test, p < 0.05) out of a total of ~1400 unique proteins were identified in the comparative spinal cord analysis (peptide FDR=0.55%).Database search and protein identification: Tandem mass spectra were extracted from .RAW files and searched using the SEQUEST-PVM v.27 (rev.9) (Eng et al., 1994) database program against a Mouse protein database downloaded as FASTA-formatted sequences from EBI-IPI (database version 3.72) which contains 56957 entries (with priority given to UniProt identifiers) as well as reverse decoy sequences to empirically assess the false identification rate. This search program was executed on a cluster computer to match the MS/MS spectra to the corresponding most highly correlated peptide sequences Mass tolerances for precursor (MS) and product ions (MS/MS) were set to 3 and 0 m/z, respectively. Searches were performed with the enzyme selectivity set to trypsin with one missed cleavage allowed and protein modifications included fixed carbamidomethylation of cysteines (57 Da). Match likelihoods were assigned a statistical confidence score using the STATQUEST probabilistic model (Kislinger et al., 2003) and candidate peptide identifications were filtered using an estimated peptide confidence score of ≥95%. A 10 ppm high accuracy mass filter accounting for isotopic shifts in the spectra was applied post-SEQUEST analysis thus improving the fidelity of protein identifications. Protein quantitation: To estimate relative protein levels, spectral counts were transformed into normalised spectral abundance factors (NSAF) as previously described (Mosley et al., 2009). Briefly, this involves dividing the spectral count (SC) of a protein by its length (Mw) and finally normalises this value to the sum of all SC/Mw.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: L Dagley

PROVIDER: PXD000255 | Pride | 2014-01-13

REPOSITORIES: Pride

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Publications

Discovery of novel disease-specific and membrane-associated candidate markers in a mouse model of multiple sclerosis.

Dagley Laura F LF Croft Nathan P NP Isserlin Ruth R Olsen Jonathan B JB Fong Vincent V Emili Andrew A Purcell Anthony W AW

Molecular & cellular proteomics : MCP 20131220 3

Multiple sclerosis is a chronic demyelinating disorder characterized by the infiltration of auto-reactive immune cells from the periphery into the central nervous system resulting in axonal injury and neuronal cell death. Experimental autoimmune encephalomyelitis represents the best characterized animal model as common clinical, histological, and immunological features are recapitulated. A label-free mass spectrometric proteomics approach was used to detect differences in protein abundance withi ...[more]

PMID: 24361864

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Project description:Tissue lysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012)) Briefly, greater than 300 mg of frozen tumor mass was homogenized and sonicated in urea lysis buffer (20 mM HEPES pH 8.0, 9 M urea, 2.5 mM sodium pyrophosphate, 1.0 mM beta-glycerophosphate, 1% N-octyl glycoside, 2 mM sodium orthovanadate). Total protein was measured using the BCA Protein Assay Kit (Thermo Scientific/Pierce) and 25 mg of total protein was used for phospho-proteomic analysis. Phospho-tyrosine peptide enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012); Rubbi, L., et al. Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling. Sci Signal 4, ra18 (2011); Graham, N.A., et al. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death. Molecular systems biology 8, 589 (2012)) Phospho-peptides were identified using the Proteome Discoverer software (version 1.3.0.339, Thermo Fisher Scientific). MS/MS fragmentation spectra were searched using SEQUEST against the Uniprot human reference proteome database with canonical and isoform sequences (downloaded January 2012 from uniprot.org). Search parameters included carbamidomethyl cysteine (*C) as a static modification. Dynamic modifications included phosphorylated tyrosine, serine, or threonine (pY, pS, pT, respectively) and oxidized methionine (*M). The Percolator node of Protein Discoverer was used to calculate false-discovery rate (FDR) thresholds and the FDR for the datasets was adjusted to 1% (version 1.17, Thermo Scientific). The Percolator algorithm uses a target-decoy database search strategy and discriminates true and false identifications with a support vector machine. The PhosphoRS 2.0 node was used to more accurately localize the phosphate on the peptide44. Only phospho-peptides with at least one phospho-tyrosine assignment with a reported probability above 20% were considered. MS2 spectra for all reported phosphopeptides are available under the PRIDE accession numbers

Project description:Nucleoids have been purified from Deinococcus deserti and Deinococcus radiodurans cells subjected to irradiation and short recovery (six replicates for each strain). The nucleoids have been analyzed by shotgun proteomics as described in Toueille M et al (2012) J Proteomics. Comparative proteomics pointed at specific proteins recruited at the nucleoids during the recovery stage after DNA damages produced by the irradiation. Data processing and bioinformatics: Peak lists were generated with the MASCOT DAEMON software (version 2.2.2) from Matrix Science using the extract_msn.exe data import filter (ThermoFisher) from the Xcalibur FT package (version 2.0.7) from ThermoFisher. Data import filter options were set at: 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1000 (threshold). Using the MASCOT search engine (version 2.2.04) from Matrix Science, we searched all MS/MS spectra against in-house polypeptide sequence databases. For Deinococcus radiodurans MS/MS assignments, the database contained i) the sequence of all currently annotated proteins coded by D. radiodurans BAA-816 genome (2629 proteins from chromosome 1 (NC_001263), 368 proteins from chromosome 2 (NC_001264), 130 proteins from plasmid MP1 (NC_000958), and 35 proteins from plasmid CP1 (NC_000958)), ii) 28 manual protein sequence curations from D. radiodurans R1, and iii) 121 additional ORF sequences predicted by the CONSORF consensus prediction system.This database thus comprises 3,311 polypeptide sequences, totaling 1,006,757 amino acids. For Deinococcus deserti MS/MS assignments, the database contained the sequence of all annotated proteins coded by D. deserti VCD115 chromosome and plasmids. This database comprises 3,455 polypeptide sequences, totaling 1,083,334 amino acids. Searches for tryptic peptides were performed with the following parameters: full-trypsin specificity, a mass tolerance of 10 ppm on the parent ion and 0.5 Da on the MS/MS, static modifications of carboxyamidomethylated Cys (+57.0215), and dynamic modifications of oxidized Met (+15.9949). The maximum number of missed cleavages was set at 1. All peptide matches with a peptide score below a stringent P value of 0.001 were filtered by the IRMa 1.18.1 parser. A protein was considered validated when at least two different peptides were detected in the same experiment. False-positives rate for protein identification was estimated using the appropriate decoy database as below 0.1% with these parameters.

Dataset Information

Label-free analysis of EAE vs Sham Spinal cord and PBMCs

Publications

Discovery of novel disease-specific and membrane-associated candidate markers in a mouse model of multiple sclerosis.

Similar Datasets

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