| Metadata |
| datasetIdentifier | PASS00031 |
| datasetType | MSMS |
| submitter | James M. Elmore <jmelmore@ucdavis.edu> |
| submitter_organization | |
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| datasetTag | Athaliana_PM_ETI |
| datasetTitle | Plasma membrane proteomics of Arabidopsis immune signaling - ETI |
| publicReleaseDate | 2012-01-10 00:00:00 |
| finalizedDate | 2012-01-10 17:32:52 |
| summary | Raw MS/MS data from a Gel LC-MS/MS experiment analyzing plasma membrane-enriched fractions isolated from 4-week old Arabidopsis plants after activation of the Resistance protein RPS2. 3 Biological Replicates. 2 conditions (treatment and control). Samples subjected to 1D SDS-PAGE and each lane cut into 15 pieces that were digested and subjected to LC-MS/MS separately (15 MS/MS runs per sample). 90 MS/MS runs total. DX=GVG-AvrRpt2 plants sprayed with Dexamethasone (treatment samples). PM=Col-0 WT plants sprayed with Dexamethasone (negative control samples). Data acquired using a Thermo Scientific LTQ ion trap mass spectrometer. |
| contributors | James Mitch Elmore, Jun Liu, Barrett Smith, Brett Phinney, Gitta Coaker |
| publication | Elmore, JM, Liu, J, Smith, B, Phinney, B, and Coaker, G. Quantitative Proteomics Reveals Dynamic Changes in the Plasma Membrane Proteome During Arabidopsis Immune Signaling. Molecular and Cellular Proteomics. In press. |
| growth | 4-week old Arabidopsis plants grown a controlled environment chamber at 24˚C with a 10h-light/14h-dark photoperiod under a light intensity of 85μE/m2/s. |
| treatment | GVG-AvrRpt2 plants and Col-0 WT plants were sprayed with dexamethasone and incubated under a light bench for 6 hours before tissue harvesting. |
| extraction | Tissue was harvested, total microsomes isolated, and then subjected to aqueous two-phase partitioning to enrich for plasma membrane vesicles. |
| separation | PM samples were delipidated, solubilized in Laemmli buffer + 6M urea, then subjected to 1D SDS-PAGE to separate proteins by size. Entire sample lanes were cut into 15 pieces. |
| digestion | Each gel lane piece was digested with trypsin according to standard protocols. |
| acquisition | The LC-MS/MS system configuration consisted of a CTC Pal autosampler (LEAP Technologies) and Paradigm HPLC (Michrom BioResources) coupled to a LTQ ion trap mass spectrometer (Thermo Scientific) with a CaptiveSpray ionization source (Michrom BioResources). Reversed phase LC was performed by injecting 60μL of each digested peptide sample onto a Zorbax300SB-C18 trap column (5μm, 5x0.3mm, Agilent Technologies) and desalted online. Peptides were then eluted from the trap and separated on a reverse-phase Michrom Magic C18AQ (200μm x 150mm) capillary column at a flow rate of 2 μL/min using a 120min gradient (2 to 35% buffer B 85min, 35 to 80% buffer B 25min, 2% buffer B 10min; buffer A=0.01% formic acid in H2O, buffer B=100% acetonitrile). The mass spectrometer was operated in data-dependent acquisition mode with 60sec dynamic exclusion enacted on masses observed twice within 30sec. A standard Top10 acquisition method was used with one survey MS scan followed by MS/MS scans on the 10 most intense ions. |
| informatics | Tandem mass spectra were extracted using Bioworks 3.3 and analyzed using the X!Tandem GPM-XE Tornado version 2009.04.01.3 spectrum modeler (30) (http://www.thegpm.org). X!Tandem was configured to search the UniProtKB Arabidopsis complete proteome sequence database (downloaded 07-07-2010, 31881 entries) and common repository of adventitious proteins database (version 1.0, 112 entries). A reversed and concatenated database served as a decoy sequence database to determine protein false discovery rate (FDR) as in (31). X!Tandem was configured to allow parent ion mass error of 1.8Da and fragment mass error of 0.4Da. Data were searched using complete carbamidomethyl modification of cysteine residues and potential modifications of oxidation of methionine and tryptophan, deamidation of asparagine and glutamine in round 1 and dioxidation of methionine and tryptophan in round 2 and allowing one missed cleavage. X!Tandem search results were imported into Scaffold 3.0.7 (Proteome Software) and all MS runs corresponding to the same sample were grouped together. Peptide identifications were modeled using the PeptideProphet algorithm (32). Protein identification FDR was determined empirically using hits from the reversed decoy database. DX=GVG-AvrRpt2 samples; PM=Col-0 WT control samples. |
| instruments | Thermo Scientific LTQ ion trap |
| species | Arabidopsis thaliana |
| massModifications | static: C+57.021464,variable: N+1, Q+1,M+16, W+16, M+32, W+32, n+42 |
