FT-ICR MS Proteomic Analysis of Endogenous TAP-tagged CTCF Transcription Factor Complexes

摘要

CTCF is an 11-zinc finger DNA-binding transcription factor with a variety of functions including gene activation and repression [1-3]. CTCF has recently been identified as a novel epigenetic regulator affecting transcription at the genome-wide level which implicates its involvement in tumorigenesis [3,4]. While many of these functions rely on CTCF-DNA interactions, protein-protein interactions also play a large role in CTCF-dependent molecular processes. This work reports on the proteomic analysis of endogenously expressed tandem-affinity-tagged CTCF protein complexes from mouse embryonic stem (ES) cells by use of FT-ICR mass spectrometry. ProtA-3XFLAG-CTCF knockin vector was constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair rescue as previously described [5]. Cell lysate from 1 X 10~(9) genetically modified (and control wild-type) mouse ES cells were incubated with anti-FLAG M2 beads (Sigma) for five hours at 4 deg C and then eluted with FLAG peptide. The 3XFLAG eluted CTCF-complexes were loaded onto a 4 to 20 percent acrylaminde gel and then subjected to whole lane GeLCMS analysis. Each was tryptically digested (with reduction and alkylation, iodoacetamide) and analyzed by use of reverse phase (RP) C_(18) liquid chromatography (LC) LTQ-FT mass spectrometry. LC-MS Results were searched against the murine FASTA database by Turbosequest (Thermo Fisher), SEQUEST, Xtandem, and Mascot followed by conversion of the various scores to probability schemes by PeptideProphet. Each search was conducted with 5 ppm precursur ion mass accuracy. The results from the three search algorithms was then combined with Scaffold.