Dataset Information

C2C12 Myoblasts and Myotubes exosomes

ABSTRACT: During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. RAW files were processed using MaxQuant [28] version 1.3.0.3. Spectra were searched against the Uniprot database (August 2012 version, Mus musculus taxonomy 10090, 86644 sequences, Bos taurus taxonomy 9913, 34280 sequences and Equus caballus taxonomy 9796, 24299 sequences) and the frequently observed contaminants database (notably containing protein sequences from serum proteins) embedded in MaxQuant. Trypsin was chosen as the enzyme and 2 missed cleavages were allowed. Precursor mass error tolerances were set respectively at 20 ppm and 6 ppm for first and main searches. Fragment mass error tolerance was set to 0.5 Da. Peptide modifications allowed during the search were: trioxidation (C, fixed), acetyl (N-ter, variable), dioxidation (M, variable), oxidation (M, variable) and deamidation (NQ, variable). Minimum peptide length was set to 7 amino acids. Minimum number of peptides, razor+unique peptides and unique peptides were set respectively to 2, 2 and1. Maximum false discovery rates - calculated by employing a reverse database strategy - were set to 0.01 at peptide and protein levels.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Equus Caballus (horse) Bos Taurus (bovine) Mus Musculus (mouse)

SUBMITTER: Yohann Couté

LAB HEAD: Yohann Couté

PROVIDER: PXD000022 | Pride | 2014-01-29

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 8

Publications

Proteomic analysis of C2C12 myoblast and myotube exosome-like vesicles: a new paradigm for myoblast-myotube cross talk?

Forterre Alexis A Jalabert Audrey A Berger Emmanuelle E Baudet Mathieu M Chikh Karim K Errazuriz Elisabeth E De Larichaudy Joffrey J Chanon Stéphanie S Weiss-Gayet Michèle M Hesse Anne-Marie AM Record Michel M Geloen Alain A Lefai Etienne E Vidal Hubert H Couté Yohann Y Rome Sophie S

PloS one 20140102 1

Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome ...[more]

PMID: 24392111

Similar Datasets

Project description:Quantitative proteomic analysis of human post-mortem substantia nigra from patients with Parkinson’s disease (n=3) versus controls (n=3) based on sixplex TMT (TMT6) isobaric labeling which allowed protein simultaneous identification and quantification. Samples were digested and pooled after TMT6 labeling. Peptides were fractionated by OFFGEL electrophoresis (24 fractions). Each peptidic fraction was analyzed in two technical replicates by LC−MS/MS analysis on a LTQ Orbitrap XL mass spectrometer equipped with a NanoAcquity HPLC system. MS data were processed using EasyProtConv (v. 1.21). Peak lists were generated from raw data combining CID and HCD spectra, and submitted to Easyprot (v2.2) which uses Phenyx (GeneBio, Geneva, Switzerland) for protein identification [1]. Searches were conducted against UniProt Swiss-Prot database (08-Feb-2011, 525’207 entries) specifying Homo sapiens taxonomy. Trypsin was selected as the proteolytic enzyme, one missed cleavage was allowed, cysteines carbamidomethylation, TMT6 amino terminus and TMT6 lysine were set as a fixed modification whereas oxidized methionine as variable. The minimum peptide length was five amino acids and precursor error tolerance was 10 ppm. False positive ratios were estimated using a reverse decoy database [3]. Peptide z-scores were set to maintain a false positive peptide ratio below 1%. Proteins with at least two unique distinct peptide sequences were selected and clustered based on shared peptides indistinguishable by MS [4] using Isobar (v 1.3.1) package. The protein entry containing the most peptides was selected as the group reporter. For protein quantification, TMT6 reporter ion intensities were extracted, corrected for isotopic impurities as provided by the manufacturer and each channel was normalized imposing equal median intensity. Only spectra from protein specific peptides not eliminated after outlier filtering were used for quantification. Isobar R package (v 1.3.1.) was used to calculate protein ratios and select statistically significant differentially regulated proteins between the two states.

Project description:Kinome profiling of human basal breast cancer cell line MDA-MB-231 using CTx-0294885 or mixture of 4 broad-spectrum kinase inhibitor (Purvalanol B, SU6668, VI16832 and CTx-0294885). Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bis-anilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high confidence phosphosites on 183 kinases, approximately 10 % of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2 and PRKDC. Therefore, CTx 0294885 represents a powerful new reagent for analysis of kinome signalling networks that may facilitate development of targeted therapeutic strategies. Data processing and bioinformatics: Raw files were processed with MaxQuant (version 1.1.1.25) for feature detection, protein identification and quantification, using the Andromeda search engine integrated into the MaxQuant environment for database searching. Extracted peak lists were searched against the UniProtKB/Swiss-Prot Homo sapiens database (Uniprot_human_2010_10) containing 35052 entries and a separate reverse decoy database for controlling the false discovery rate (FDR). The following search parameters were selected; fixed cysteine carbamidomethylation modification; variable methionine oxidation modification, variable protein N-acetylation, variable phosphorylation of serine, threonine and tyrosine; minimum peptide length of 6 amino acids and up to 2 missed cleavages were allowed. In addition, for SILAC experiments, the SILAC labels Arg10 and Lys8 were selected as modifications, and minimum peptide count for protein quantification was set to 1. The initial first search mass tolerance was 20 ppm for precursor ions and 0.5 Da for fragment ions, with individualized peptide mass tolerances used for the subsequent searches. The ‘match between runs’ option in MaxQuant was used to transfer identifications between runs based on matching of precursors with high mass accuracy. The FDR was limited to 1 % for both protein and peptide identifications. Peptides with posterior error probability greater than 10 % were removed and protein identification required a minimum of 1 unique peptide. For phosphopeptides, those exhibiting a phosphosite localization probability (LP) > 0.75 were included in further analyses.

			Action	DRS
	MB-1.RAW	Raw
	MB-2.RAW	Raw
	MB-3.RAW	Raw
	MT-1.RAW	Raw
	MT-2.RAW	Raw