Dataset Information

Identification of telomere-binding proteins by enChIP-MS

ABSTRACT: We identified the telomere associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitaion (enChIP). Purified proteins were analyzed by GeLC-MS/MS. Tandem mass spectra were extracted by Proteome Discoverer 1.2. Database searchs were submitted to an in-house Mascot server version 2.4.1. Mascot was set up to search the Swissprot database assuming the digestion enzyme as trypsin. Carbamidomethylation of cysteine was set as a fixed modification. Oxidized methionine, acetylation of N terminus and conversion of glutamine to Pyro-Glu at N terminus were set as variable modifications. The precursor mass tolerances were 10 ppm and the tolerance of the MS/MS ions was 0.8 Da. In Scaffold 3.4.5, database search results were grouped according to gel bands.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Ba/f3 Cell

SUBMITTER: Kazunobu Saito

LAB HEAD: Kazunobu Saito

PROVIDER: PXD000461 | Pride | 2013-11-12

REPOSITORIES: Pride

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Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).

Fujita Toshitsugu T Asano Yoshinori Y Ohtsuka Junko J Takada Yoko Y Saito Kazunobu K Ohki Rieko R Fujii Hodaka H

Scientific reports 20131108

Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-b ...[more]

PMID: 24201379

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Project description:The use of internal calibrants (the so called lock mass approach) provides much greater accuracy in mass spectrometry based proteomics. However, the polydimethylcyclosiloxane (PCM) peaks commonly used for this purpose are quite unreliable, leading to missing calibrant peaks in spectra and correspondingly lower mass measurement accuracy. Therefore, we here introduce a universally applicable and robust internal calibrant, the tripeptide Asn3. We show that Asn3 is a substantial improvement over PCM both in terms of consistent detection and resulting mass measurement accuracy. Asn3 is also very easy to adopt in the lab, as it requires only minor adjustments to the analytical setup. Data analysis: For mass measurement accuracy (MMA) calculations and comparisons, the following Mascot workflow was used. From the MS/MS data in each LC run, Mascot Generic Files were created using Distiller software (version 2.4.3.3, Matrix Science, London, UK, www.matrixscience.com/distiller.html). These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.4.0, Matrix Science). Spectra were searched against the Swiss-Prot database (version 13_04 of UniProtKB/Swiss-Prot protein database containing 20,232 sequence entries of human proteins) concatenated with its reversed sequence database. Variable modifications were set to pyro-glutamate formation of amino terminal glutamine and acetylation of the protein N-terminus, whereas fixed modifications only included oxidation of methionine. Mass tolerance on peptide ions was set to 10 ppm (with Mascot’s C13 option set to 1), and the mass tolerance on peptide fragment ions was set to 20 millimass units (mmu), except for the space-charge effect experiment(LMA5) where an extra search was done with a setting of 3 mmu. The peptide charge was set to 1+,2+,3+ and instrument setting was put on ESI-QUAD. Enzyme was set to trypsin allowing for one missed cleavage, and cleavage was allowed when arginine or lysine is followed by proline. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. All data was processed and managed by ms_lims.

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