PXD008044

PXD008044 is an original dataset announced via ProteomeXchange.

Title	MudPIT analyses of the proteins purified by in vitro dCas9-targeted chromatin IP of telomere sequences in HeLa cells
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).
HostingRepository	MassIVE
AnnounceDate	2018-02-21
AnnouncementXML	Submission_2018-02-21_06:33:58.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Laurence Florens
SpeciesList	scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine
Instrument	LTQ; LTQ XL

Revision	Datetime	Status	ChangeLog Entry
0	2017-10-25 09:36:41	ID requested
⏵ 1	2018-02-21 06:33:59	announced

no publication

submitter keyword: Telomere repeat sequences, Caspase 9, reverse chromatin immunoprecipitation, protein:RNA interaction, HeLa

Laurence Florens
contact affiliation	The Stowers Institute for Medical Research
contact email	laf@stowers.org
lab head
Laurence Florens
contact affiliation	Stowers Institute for Medical Research
contact email	laf@stowers.org
dataset submitter

MassIVE dataset URI

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