Project PXD000280

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Summary

Title

Arabidopsis thaliana Cytochrome c Novel Partners Identification by LTQ

Description

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.

Sample Processing Protocol

See details in reference PMID : 24019145

Data Processing Protocol

See details in reference PMID : 24019145

Contact

Jonathan Martinez Fabregas, Cell Signaling and Immunology

Submission Date

10/06/2013

Publication Date

11/11/2013

Cell Type

plant cell

Instrument

LTQ

Quantification

Not available

Experiment Type

Bottom-up proteomics

Assay count

8

Publication

    Martínez-Fábregas J, Díaz-Moreno I, González-Arzola K, Janocha S, Navarro JA, Hervás M, Bernhardt R, Díaz-Quintana A, De la Rosa MÁ; New Arabidopsis thaliana Cytochrome c Partners: A Look Into the Elusive Role of Cytochrome c in Programmed Cell Death in Plants., Mol Cell Proteomics, 2013 Dec, 12, 12, 3666-76, PubMed: 24019145

Assay

Showing 1 - 8 of 8 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 29628 A111C Cytochrome c control 25 72 26 3235 69
2 29629 A111C Cytochrome c Control 2 hours 27 89 28 4688 83
3 29630 A111C Cytochrome c Control 2 hours ok 31 129 33 4907 120
4 29631 Blank Column Control 10 41 12 5067 39
5 29632 A111C Cytochrome c H2O2 39 126 52 3618 115
6 29633 A111C Cytochrome c H2O2 2 hours 63 352 65 5368 260
7 29634 A111C Cytochrome c H2O2 2 hours ok 62 208 69 4806 148
8 29635 Blank column H2O2 39 143 43 5494 126