PRIDE Assigned Tags:Biomedical Dataset
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Identification of proteins stably associated with G9a/GLP (EHMT2/EHMT1) complex of histone methyltransferases
G9a (EHMT2) and the G9a-like protein GLP (EHMT1) form a stable G9a/GLP heterodimer in embryonic stem cells and function cooperatively to establish and maintain the abundant repressive H3K9me2 modification, in addition to modifying several non-histone proteins. The G9a-dependent H3K9me2 is implicated in lineage-specific gene silencing and covers large chromosomal domains. While the mechanism of H3K9me2maintenance by G9a/GLP is known, how new patterns of this modification are established is not well understood. With this in mind, we used FLAG affinity purification of G9a under two different stringency conditions (150 and 300 mM NaCl) coupled with mass spectrometry to identify proteins stably associated with G9a/GLP, which could serve as potential recruiters of the complex to unmodified chromatin.
Sample Processing Protocol
750 ug of total nuclear protein extracted from mouse ES cells, wild type and expressing N-terminally FLAG-tagged G9a, with buffer containing either 150 or 300 mM NaCl were incubated with 25 ul FLAG® M2 agarose beads (Sigma). The beads were washed 4 times with buffer containing either 200 mM NaCl and the bound proteins were eluted with FLAG peptide (Sigma). Protein samples were boiled with loading buffer and mercaptoethanol for 5 min and the were seprated by with an SDS-PAGE gel (Novex) for 10mins (200V). The gel was stained with Imperial stain for 1 hour and destained overnight. Stained band were exiced and the proteins were subjected to tryptic digestion as previously described (Shevchenko et al, 1996). In brief, proteins were reduced in 10 mM dithiothreitol (Sigma Aldrich, UK) for 30 min at 37°C and alkylated in 55 mM iodoacetamide (Sigma Aldrich, UK) for 20 min at ambient temperature in the dark. They were then digested 16 hours at 37°C with 12.5 ng μL-1 trypsin (Pierce, UK). After digestion, samples were diluted with equal volume of 0.1% TFA and spun onto StageTips as describe by Rappsilber et al (2003). Peptides were eluted in 20 μL of 80% acetonitrile in 0.1% TFA and concentrated down to 4 μL by vacuum centrifugation (Concentrator 5301, Eppendorf, UK). The peptide sample was then prepared for LC-MS/MS analysis by diluting it to 5 μL by 0.1% TFA. MS anaysis was performed on a Velos LTQ Orbitrap (THermo Fisher Scientific) coupled on-line, to Ultimate 3000 RSLCnano Systems (Dionex, Thermo Fisher Scientific). The analytical column with a self-assembled particle frit (Ishihama et al 2002) and C18 material (ReproSil-Pur C18-AQ 3 μm; Dr. Maisch, GmbH, Germany) was packed into a spray emitter (75-μm ID, 8-μm opening, 300-mm length; New Objective) using an air-pressure pump (Proxeon Biosystems, USA). Mobile phase A consisted of water and 0.1% formic acid; mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. The gradient t used 130 minutes. The peptides were loaded onto the column at a flow rate of 0.5 μL min-1 and eluted at a flow rate of 0.2 μL min-1 according to the following gradient: 2 to 40% buffer B in 90 min, then to 95% in 21 min. THe scan range was set between 300 and 1700 m/z and FTMS spectra were recorded at 60,000 resolution. The twenty most intense peaks with charge ≥ 2 of the MS scan were selected with an isolation window of 2.0 Thomson in the ion trap for MS2 (normal scan, wideband activation, filling 5.0E5 ions for MS scan, 1.0E4 ions for MS2, maximum fill time 100 ms, dynamic exclusion for 60 s).
Data Processing Protocol
Searches were conducted using MASCOT (ver. 2.4.1) against a database containing Mus musculus sequences (complete/reference proteome set of UniProt database). The search parameters were: MS accuracy, 6 ppm; MS/MS accuracy, 0.6 Da; enzyme, trypsin; allowed number of missed cleavages, 2; fixed modification, carbamidometylation on cysteine; variable modification, oxidation on methionine.
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||61042||Iteracting partners of the G9a protein||173||919||543||14504||617||
|2||61041||Interacting Partners of G9a||413||1953||1327||14955||1435||
|3||61039||Interacting Partners of the G9a protein||197||1119||609||15102||677||
|4||61040||Intercting Partners of the G9a protein||420||2004||1378||15547||1475||