Dataset Information

In vitro reconstitution of epigenetic reprogramming in the human germ line

ABSTRACT: M1-BTAG iPSCs or TET1-KO iPSCs were analyzed by 8% SDS-PAGE, followed by trypsin digestion and LC/MS/MS with an Orbitrap Fusion Lumos linked with an Ultimate 3000 pump and HTC-PAL autosampler. Acquired data files were processed using FragPipe v20.0 (MSFragger v3.8, Philosopher v5.0.0, IonQuant v1.9.8) for protein identification and quantitation. Peptides and proteins were identified by automated database searching against the human UniprotKB/Swissprot database (accessed on 2021/07/15) with settings of strict trypsin (C-terminal of K and R) specificity and the allowance for up to 2 missed cleavages. Carbamidomethylation of cysteine (C, +57.021465) was set as a fixed modification. Oxidation of methionine (M, +15.9949) and acetylation of protein N-terminus (Protein N-term, +42.0106) were set as variable modifications. A total of 2139 protein groups were identified.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Mitinori Saitou

PROVIDER: PXD048118 | JPOST Repository | Thu Apr 25 00:00:00 BST 2024

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	231109ko02E_KO1_1H.raw	Raw
	231109ko02E_KO1_1H_uncalibrated.mzML	Mzml
	231109ko03F_KO1_1L.raw	Raw
	231109ko03F_KO1_1L_uncalibrated.mzML	Mzml
	231109ko04G_KO1_2H.raw	Raw

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Publications

In vitro reconstitution of epigenetic reprogramming in the human germ line.

Murase Yusuke Y Yokogawa Ryuta R Yabuta Yukihiro Y Nagano Masahiro M Katou Yoshitaka Y Mizuyama Manami M Kitamura Ayaka A Puangsricharoen Pimpitcha P Yamashiro Chika C Hu Bo B Mizuta Ken K Tsujimura Taro T Yamamoto Takuya T Ogata Kosuke K Ishihama Yasushi Y Saitou Mitinori M

Nature 20240520 8019

Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency<sup>1</sup>. In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitoti ...[more]

PMID: 38768632

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Project description:Before stimulation, the cells were washed and the culture media was changed to RPMI-1640. After stimulation, cell culture media were collected, concentrated, and the proteins were precipitated with 2-D Clean-Up Kit (GE Healthcare) followed by protein alkylation, trypsin digestion and iTRAQ labelling of the resulting peptides according to manufacturer´s instructions (Applied Biosystems). Control sample was labeled with 114, LPS-stimulated with 115, Curdlan-stimulated with 116, and GBY-stimulated with 117 isobaric tag. After labelling the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing labelled peptides was analyzed twice with nano-liquid chromatography-tandem mass spectrometry using Ultimate 3000 nano-liquid chromatograph (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems / MDS Sciex) with nano-ESI ionization as previously described (Lietzén et al., 2011). MS data were acquired automatically using Analyst QS 2.0 software. Protein identification and relative quantitation were performed with ParagonTM search algorithm (Shilov et al., 2007) using ProteinPilot 2.0 interface (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. Database searching was done against UniProt human database (version 2008-01-28 with 20330 human sequences) and ´decoy` database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). Importantly, automatic bias correction was not used in quantitation. The false discovery rates were calculated as previously described (Elias and Gygi, 2007) and were 1.4% and 2% for the two biological replicates.