Dataset Information

Telomeric protein composition deriving from q-TIP analysis comparing Hela supertelomerase cells, Hela TRF2 knockdown cells or Hela POT1 knockdown cells with Hela control cells, proteomes by 2D-LC-MS/MS

ABSTRACT: All samples derived from the q-TIP experiments were analysed using reverse-phase chromatography coupled to orbitrap high-resolution mass spectrometer using trypsin digestion. MS/MS files were searched against the UniProt human protein database (release date 18-04-2012) by MaxQuant software (version 1.2.2.5 and 1.3.0.5). Mass spectra were searched with an initial mass tolerance of 7 ppm in MS mode and 0.5 Da in MS/MS mode. Up to two missed cleavages were allowed. Carbamidomethylation was set as a fixed modification, whereas oxidation (M) and acetylation (Protein N-term) were considered as variable modifications. Phospho (STY) was set as variable modifications for the approach B as well. Minimum required peptide length was set to six amino acids (seven for the approach B) and at least two (unique + razor) peptides were required for protein identification. A cut-off was fixed at 1% FDR at the peptide and protein level. Reverse and contaminants sequences were removed and proteins with a Posterior Error Probability (PEP) lower than 0.1 were accepted for further data treatment. Protein quantification was performed with razor and unique peptides, using only unmodified, oxidated (M) and acetylated (Protein N-term) peptides. A minimum of two ratio counts was required to quantify proteins. A FDR < 0.05 was applied on the Significances B to accept proteins significantly differentially quantified between conditions. In the article and the supplementary table 2, Rep3 corresponds to the forward experiment or the second replicate (Rep2)

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Romain Hamelin

PROVIDER: PXD000243 | Pride | 2013-11-26

REPOSITORIES: Pride

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Publications

A quantitative telomeric chromatin isolation protocol identifies different telomeric states.

Grolimund Larissa L Aeby Eric E Hamelin Romain R Armand Florence F Chiappe Diego D Moniatte Marc M Lingner Joachim J

Nature communications 20130101

Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we speci ...[more]

PMID: 24270157

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Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.

Project description:1D-LC-MS/MS analysis of OGE-prefractionated and TMT labeled mouse samples consiting of 2 unstimulated and 2 stimulated T-cell samples. The acquired raw-files were converted to the mascot generic file (mgf) format using the msconvert tool (part of ProteoWizard, version 3.0.4624 (2013-6-3)). Using the MASCOT algorithm (Matrix Science, Version 2.4.0), the mgf files were searched against a decoy database containing normal and reverse sequences of the predicted SwissProt entries of mus musculus (www.ebi.ac.uk, release date 16/05/2012) and commonly observed contaminants (in total 33,832 sequences) generated using the SequenceReverser tool from the MaxQuant software (Version 1.0.13.13). The precursor ion tolerance was set to 10 ppm and fragment ion tolerance was set to 0.01 Da. The search criteria were set as follows: full tryptic specificity was required (cleavage after lysine or arginine residues unless followed by proline), 2 missed cleavages were allowed, carbamidomethylation (C), TMT6plex (K and peptide n-terminus) were set as fixed modification and oxidation (M) as a variable modification. Next, the database search results were imported to the Scaffold Q+ software (version 4.1.1, Proteome Software Inc., Portland, OR) and the protein false identification rate was set to 1% based on the number of decoy hits. Specifically, peptide identifications were accepted if they could be established at greater than 94.0% probability to achieve an FDR less than 1.0% by the scaffold local FDR algorithm. Protein identifications were accepted if they could be established at greater than 6.0% probability to achieve an FDR less than 1.0% and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet program (Nesvizhskii, et al, Anal. Chem. 2003; 75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. For quantification, acquired reporter ion intensities in the experiment were globally normalized across all acquisition runs. Individual quantitative samples were normalized within each acquisition run. Intensities for each peptide identification were normalized within the assigned protein. The reference channels were normalized to produce a 1:1 fold change. All normalization calculations were performed using medians to multiplicatively normalize data. A list of identified and quantified proteins is available in the xls file.

Dataset Information

Telomeric protein composition deriving from q-TIP analysis comparing Hela supertelomerase cells, Hela TRF2 knockdown cells or Hela POT1 knockdown cells with Hela control cells, proteomes by 2D-LC-MS/MS

Publications

A quantitative telomeric chromatin isolation protocol identifies different telomeric states.

Similar Datasets

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