PXD000143

PXD000143 is an original dataset announced via ProteomeXchange.

Title	Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives
Description	Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC) and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC) and adult mouse brain tissue. Readers for these modifications are only partially overlapping and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation. Peptides were separated on an EASY-nLC (Proxeon) connected online to an LTQ-Orbitrap-Velos mass spectrometer. Spectra were recorded in CID mode. A gradient of organic solvent (5-30% acetonitrile) was applied (120 minutes) and the top 15 most abundant peptides were fragmented for MS/MS, using an exclusion list of 500 proteins for 45 seconds. Raw data were analyzed using Maxquant version 1.2.2.5 and the integrated Andromeda search engine against protein database ipi.MOUSE.v3.68. Using Perseus, data was filtered for contaminants, reverse hits, number of peptides (>1) and unique peptides (>0). Ratios were logarithmitized (log2) and groups (consisting of forward and reverse) were defined. Proteins were filtered to have at least 2 valid values in one of the groups and missing values were imputed based on a normal distribution (width=0.2 and shift=0), after which Significance B was calculated (Benj.Hoch.FDR=0.05). Scatterplots were made using R. Proteins were defined to be significant when both forward and reverse significance p<0.05 and minimal ratios were >2 in both experiments. The H/L ratios shown in Figure2A-C were calculated using the formula (log(forward ratio) - log(reverse ratio))/2.
HostingRepository	PRIDE
AnnounceDate	2013-02-26
AnnouncementXML	Submission_2013-02-26_03:36:31.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
Reprocessed datasets that are derived from this dataset	RPXD000667
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	A.H. Smits
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	No PTMs are included in the dataset
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-02-08 09:16:07	ID requested
1	2013-02-26 03:29:13	announced
1	2014-07-25 00:52:42	announced
⏵ 2	2013-02-26 03:36:31	announced	Add reference

Spruijt CG, Gnerlich F, Smits AH, Pfaffeneder T, Jansen PW, Bauer C, M, ü, nzel M, Wagner M, M, ü, ller M, Khan F, Eberl HC, Mensinga A, Brinkman AB, Lephikov K, M, ü, ller U, Walter J, Boelens R, van Ingen H, Leonhardt H, Carell T, Vermeulen M, Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell, 152(5):1146-59(2013) [pubmed]

submitter keyword: 5-hydroxymethylcytosine, stem cells, differentiation, quantitative mass spectrometry, chromatin readers

A.H. Smits
contact affiliation	University Medical Center Utrecht
contact email	a.smits-4@umcutrecht.nl

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