Dataset Information

TAILS with Arabidopsis thaliana wilde typ, tic56-1and ppi2

ABSTRACT: Aim was to analyse the in vivo start position of proteins in the import-mutant plant lines tic56-1 and ppi2 in contrast to wild type. Therefor we enriched N-terminal peptides originating from total protein of the three plant lines and detect them by mass spectrometry. This method is called TAILS and was described by Doucet et al. 2011 previously. For this analysis two experiments were done, further called TAILS1 and TAILS2. In TAILS1 total soluble protein from wild type and tic56-1 were analysed. N-termini of the wild type were labelled using normal formaldehyde during N-termini from tic56-1 were labelled with heavy formaldehyde. After labelling the two samples were combined and afterward treaded and measured together. In TAILS2 also membrane proteins were solubilized and additionally total protein from ppi2 plants was analysed. In this experiment the protein samples were treaded in the whole procedure separately and all three samples were labelled using normal formaldehyde. For peptide identification the raw-data were analysed using the Proteome Discoverer (search engine: SEQUEST) as well as MaxQuant (search engine: Andromeda). As variable modifications N-acetylation and methionine oxidation and as fixed modification carbamidomethylation of cysteins and dimethylation of lysine residues and N-termini (TAILS1: normal for wild type and heavy for tic56-1 sample) were allowed. For the Proteome Discoverer analysis the peptides diverse in the isotope labels (TAILS1) or the blocked N termini (N-terminal acetylation or dimethylation) were identified in distinct searches.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Sacha Baginsky

LAB HEAD: Sacha Baginsky

PROVIDER: PXD000660 | Pride | 2015-02-03

REPOSITORIES: Pride

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Publications

Characterization of chloroplast protein import without Tic56, a component of the 1-megadalton translocon at the inner envelope membrane of chloroplasts.

Köhler Daniel D Montandon Cyril C Hause Gerd G Majovsky Petra P Kessler Felix F Baginsky Sacha S Agne Birgit B

Plant physiology 20150301 3

We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC su ...[more]

PMID: 25588737

Similar Datasets

Project description:Pilotexp_IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_FT_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The unphosphorylated peptides found in the flowthrough of the TiO2 column were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. The iTRAQ ratio obtained for the unmodified peptides from this flowthrough sample was used to calculate protein ratios for the 4 different conditions.

			Action	DRS
	TAILS1.raw	Raw
	TAILS1_N-acetyl_tic56-1.msf	Msf
	TAILS1_N-acetyl_wt.msf	Msf
	TAILS1_dimethyl_tic56-1.msf	Msf
	TAILS1_dimethyl_wt.msf	Msf