Dataset Information

Cytosol - Analysis of the Arabidopsis cytosolic proteome highlights subcellular partitioning of central plant metabolism

ABSTRACT: Enriched Arabidopsis cytosol (cell culture) fractionated by SCX (15 fractions) and analysed by Q-TOF nanoLC-MS/MS (AB Sciex QStar Elite)

INSTRUMENT(S): instrument model, QSTAR

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

SUBMITTER: Joshua Heazlewood

PROVIDER: PRD000391 | Pride | 2011-03-01

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_16503.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_16503.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_16504.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_16504.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_16505.pride.mgf.gz	Mgf

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Publications

Analysis of the Arabidopsis cytosolic proteome highlights subcellular partitioning of central plant metabolism.

Ito Jun J Batth Tanveer S TS Petzold Christopher J CJ Redding-Johanson Alyssa M AM Mukhopadhyay Aindrila A Verboom Robert R Meyer Etienne H EH Millar A Harvey AH Heazlewood Joshua L JL

Journal of proteome research 20110228 4

The plant cell cytosol is a dynamic and complex intracellular matrix that, by definition, contains no compartmentalization. Nonetheless, it maintains a wide variety of biochemical networks and often links metabolic pathways across multiple organelles. There have been numerous detailed proteomic studies of organelles in the model plant Arabidopsis thaliana, although no such analysis has been undertaken on the cytosol. The cytosolic protein fraction from cell suspensions of Arabidopsis thaliana wa ...[more]

PMID: 21166475

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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.