Dataset Information

MudPIT analyses of the proteins purified by in vitro dCas9-targeted chromatin IP of the fruit fly Histone Cluster

ABSTRACT: 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 have applied this dCas9-based system to identify novel factors involved in D. melanogaster histone cluster (HisC) gene expression. Chromatin-associated proteins were purified by dCas9-HisC revChIP (using eight different sgRNAs of H2A and H2B genomic targets) 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. 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 ProLuCID (v. 1.3.3) (Xu et al., 2015) against a D. melanogaster non-redundant protein database (NCBI, 02-20-2013) containing 160 usual contaminants (human keratins, IgGs, and proteolytic enzymes). 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. 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).

INSTRUMENT(S): LTQ, LTQ XL

ORGANISM(S): Drosophila Melanogaster (ncbitaxon:7227)

SUBMITTER: Laurence Florens

PROVIDER: MSV000081643 | MassIVE | Wed Oct 25 09:13:00 BST 2017

SECONDARY ACCESSION(S): PXD008043

REPOSITORIES: MassIVE

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Publications

dCas9-targeted locus-specific protein isolation method identifies histone gene regulators.

Tsui Chiahao C Inouye Carla C Levy Michaella M Lu Andrew A Florens Laurence L Washburn Michael P MP Tjian Robert R

Proceedings of the National Academy of Sciences of the United States of America 20180305 12

Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromat ...[more]

PMID: 29507191

Similar Datasets

Project 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).

Project description:Fly embryos (w1118, Dlg-GFP, or Scrib-GFP) were lysed with lysis buffer (25mM Tris-HCl, 150mM EDTA, 1% NP-40, 5% glycerol, 1mM DTT, 1x protease inhibitor). Debris was removed by centrifugation, and extracts were cleared by incubation with Dynabeads (1 hour). Immunocomplexes were formed by incubation for 2 hours with Dlg antibody-conjugated magnetic beads or GFP-nanobody (GFP-Trap_MA, ChromoTek). Immunocomplexes were washed with wash buffer (25mM Tris-HCl, 150mM EDTA), eluted with elution buffer (50mM glycine PH 2.5, 150mM NaCl), and then neutralized with Tris-HCl PH 9.5. Two biological replicates of Dlg-IPs (and their corresponding negative controls without antibody) and two GFP-IPs from flies expressing either Dlg-GFP or Scrib-GFP (and their negative GFP-IP control) were analyzed by MudPIT mass spectrometry. In brief, TCA-precipitated protein eluates were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. 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 (Eng et al., 1994) against a D. melanogaster database (NCBI, 2012-03-08 release) containing 18,564 non-redundant proteins and 177 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized and searched at the same time. 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, 2.5 for doubly, and 3.5 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic. The FDRs were 0.5 +/- 0.3 and 1.3 +/- 0.8 at the peptide and protein levels, respectively. 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).