Dataset Information

Arabidopsis thaliana Cytochrome c Novel Partners Identification by LTQ

ABSTRACT: Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, it is necessary for development and for the hypersensitive response to stress or pathogenic infection. A common feature to programmed cell death among organisms is the mitochondria-to-cytosol translocation of cytochrome c. To understand the role of cytochrome c in the onset of plant programmed cell death, a proteomic approach based on affinity chromatography has been developed, using Arabidopsis cytochrome c as bait. Eleven putative, new cytochrome c partners have been identified. Nine of them bind the heme protein in plant protoplasts and in human cells, as a heterologous system, according to Bimolecular Fluorescence Complementation. The binding affinities and the kinetic rate constants of three cytochrome c - target complexes have been estimated by Surface Plasmon Resonance. Our data suggest that the role of cytochrome c as a programmed cell death-signaling messenger could be evolutionarily well-conserved. Data Processing & data analysis: Peptides were analyzed using a nanoliquid chromatography-MS/MS on an LTQ linear ion trap mass spectrometer (Thermo Electron San Jose, CA, USA). The mass spectrometer was operated in the data-dependent mode to automatically switch between full MS and MS/MS acquisition. The parameters for ion scanning were the following: Full-scan MS(400-1800 m/z) plus top seven peaks Zoom/MS/MS (isolation width 2 m/z), normalized collision energy 35%. The scanning was performed using a dynamic exclusion list (120s exclusion list size of 50). Peak lists from all MS/MS spectra were extracted from the Xcalibur RAW files using a freely available program DTAsupercharge v.1.19 (http://msquant.sourceforge.net/). MASCOT 2.1 was used to search the Uniprot_Arabidopsis protein database 100323 (90895 sequences). Database search parameters used were the following: trypsin as enzyme; peptide tolerance, 300ppm; fragment ion tolerance, 0.6 Da; missed cleavage sites,1; fixed modification, carbamidomethyl cysteine and variable modifications, methionine oxidation. In all protein identifications the probability scores were greater than the score fixed by MASCOT (30) as significant with a p-value <0.05.

INSTRUMENT(S): instrument model, LTQ

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

TISSUE(S): Plant Cell, Cell Suspension Culture

SUBMITTER: Jonathan Martinez Fabregas

PROVIDER: PXD000280 | Pride | 2013-11-11

REPOSITORIES: Pride

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Publications

New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

Martínez-Fábregas Jonathan J Díaz-Moreno Irene I González-Arzola Katiuska K Janocha Simon S Navarro José A JA Hervás Manuel M Bernhardt Rita R Díaz-Quintana Antonio A De la Rosa Miguel Á MÁ

Molecular & cellular proteomics : MCP 20130909 12

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on ...[more]

PMID: 24019145

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Project description:Using a discovery proteomics approach, the expressed proteome of Bartonella henselae, a Gram-negative prokaryotic model organism was exhaustively studied under two conditions that mimic those encountered in different hosts. Using the analysis-driven experimentation (ADE) feedback-loop strategy, we were able to virtually eliminate the biases of commonly under-represented short, basic, and particularly lower abundant and membrane protein classes, all of which are experimentally tractable. Based on a very stringent FDR at the PSM level, we identified 85% of all distinct, annotated proteins and above 90% compared to the expressed protein-coding genes in the two conditions. Several lines of evidence indicated that this is very close to all proteins that can be identified by a discovery proteomics approach with current technology Our experimental strategy relied on four elements: First, we used a combination of sub-cellular fractionation (Cyt, TM, IM, OM) with additional biochemical fractionation regimens (gelfiltration, OGEpep, OGEprot, ProteoMiner) to reduce the overall sample complexity. Second, an exclusion list approach was applied, where each sample is measured twice in a mass spectrometer. Third, we relied on the ADE strategy to target typically under-represented areas of the proteome. Finally, we also used chymotrypsin as enzyme in addition to trypsin for all membrane-derived fractions. For computational analysis, we combined results from two database search engines (Mascot and MS-GF+).Mass spectra were searched against a protein sequence database containing 1488 NCBI RefSeq annotated B. henselae proteins (NC_005956.1), 3336 sheep proteins, a positive control (myc-gfp), as well as protein sequences of 256 common contaminants. Spectra were searched against this database using the target/decoy option either with Mascot (version 2.3.0, Matrix Science) or with MS-GF+ (MS-GFDB v7747, kindly provided by Dr. Sangtae Kim, UCSD, USA) using the following parameters: Carbamidomethylation was set as a fixed modification on all Cysteines, oxidation of Methionines, deamidation of Asparagines and Glutamines, as well as cyclization of N-terminal Glutamines were considered as optional modifications. Spectra were searched for a match to fully-tryptic and semi-tryptic peptides with up to two missed cleavage sites. Precursor ion mass tolerance was set to 5 ppm, fragment ion mass tolerance was set to 0.8 Da, and the automatic decoy search option was enabled. For Mascot, data were further post-processed with Percolator (Brosch et al. 2009). Additional RNA-seq data are at GEO under the accession: GSE44564.

Project description:Abstract still has to be written. The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 um internal diameter (I.D.) x 20 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 um I.D. x 150 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A' (0.05% formic acid) to 55% solvent B' (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B'. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the human (H. sapiens) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2).

Dataset Information

Arabidopsis thaliana Cytochrome c Novel Partners Identification by LTQ

Publications

New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

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