MassIVE MSV000081645

Partial Public PXD008044

MudPIT analyses of the proteins purified by in vitro dCas9-targeted chromatin IP of telomere sequences in HeLa cells

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Description

We used a reverse chromatin immunoprecipitation (ChIP) method that takes advantage of the RNA-mediated DNA-targeting capability of Cas9 to efficiently and adaptably isolate specific genomic regions and their associated protein factors. By using purified recombinant catalytically-dead Cas9 (dCas9) combined with single-guide RNAs (sgRNAs) to form specific ribonucleoprotein (RNP) complexes, our method does not require specialized cell lines and can be easily modified to target multiple loci in any cell line or tissue. We first validated the method by targeting telomere sequences in HeLa cells. These sequences are relatively abundant, comprising 0.01% to 0.07% of the genome, and the major proteins that bind them are known. Telomere-associated proteins were purified by dCas9-Telo revChIP and were analyzed by MudPIT mass spectrometry. A control dCas9 revChIP using non-specific sgRNAs was analyzed in parallel. In brief, TCA-precipitated protein eluates were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. One sample was digested with endoproteinase AspN followed by GluC to improve identification of the TPP1 and RIF1 proteins. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on linear ion traps. MS/MS datasets were searched using SEQUEST (v. 27.9) against a non-redudant human protein database (NCBI, 03-25-2015) containing 160 usual contaminants and the dCas9 protein sequence. To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized. Peptide/spectrum matches were sorted and selected using DTASelect (Tabb et al., 2002) with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic (except for the AspNGluC-digested samples). Peptide hits from multiple runs were compared using CONTRAST (Tabb et al., 2002). The distributed normalized spectral abundance factors (dNSAF) were used to estimate relative protein levels (Zhang et al., 2010). [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Telomere repeat sequences ; Caspase 9 ; reverse chromatin immunoprecipitation ; protein:RNA interaction ; HeLa

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Tsui C, Inouye C, Levy M, Lu A, Florens L, Washburn MP, Tjian R.
dCas9-targeted locus-specific protein isolation method identifies histone gene regulators.
Proc. Natl. Acad. Sci. U.S.A. 2018 Mar 20;115(12):E2734-E2741. Epub 2018 Mar 5.

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