Project : PXD000160

View in PRIDE Inspector
Download Project Files

Summary

Title

Soybean root tip proteins under flooding with ABA

Description

Flooding is a serious problem for soybean cultivation because it markedly reduces growth. We found that treatment with ABA enhances survival ratio of soybean under flooding stress. To investigate the role of ABA in soybean under flooding stress, proteomic changes were analyzed. Total protein and nuclear protein fractions from root tips of soybean seedlings that treated with flooding or flooding with ABA were analyzed. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin and lysyl endopeptidase. Resulting peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Using an Ultimate 3,000 nanoLC system (Dionex), peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and their separation and spraying were done using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3, Nikkyo Technos) with a spray voltage of 1.5 kV. The elution was done with a linear acetonitrile gradient 8-30% in 120 min for gel free proteomics in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over a mass range of 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy. The top 10 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 sec to prevent repetitive selection of peptides.Identification of proteins were performed by Mascot search engine (version 2.3.0.2, Matrix Science) using soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0)and a common contaminants database (262 sequences). Parameters used in Mascot searches were follows: Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charge was set at +2, +3, and +4. Automatic decoy database search was performed in the search. Mascot results were filtered with Mascot percolator to improve accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were less than 1.0%. The Mascot results were used for differential analysis using SIEVE software (version 2.0, Thermo Fisher Scientific).

Sample Processing Protocol

See details in reference PMID : 23808807

Data Processing Protocol

See details in reference PMID : 23808807

Contact

Yohei Nanjo, Institute of Crop Science

Submission Date

26/02/2013

Cell Type

plant cell

Quantification

Not available

Experiment Type

Bottom-up proteomics

Assay count

24

Publication

    Komatsu S, Han C, Nanjo Y, Altaf-Un-Nahar M, Wang K, He D, Yang P; Label-free quantitative proteomic analysis of abscisic acid effect in early-stage soybean under flooding., J Proteome Res, 2013 Nov 1, 12, 11, 4769-84, PubMed(s) : 23808807

Assay

Accession Title Protein Count Peptide Count Unique Peptide Count Spectrum Count Identified Spectrum Count
28646 Soybean seedling Flood + ABA treatment Nuclear2d1 1519 13389 3296 18645 7660
28647 Soybean seedling Flood + ABA treatment Nuclear2d2 1818 14759 4103 19555 9051
28648 Soybean seedling Flood + ABA treatment Nuclear2d3 1848 14037 4282 19366 9108
28649 Soybean seedling Flood + ABA treatment Nuclear4d1 1661 12957 3710 18577 8091
28650 Soybean seedling Flood + ABA treatment Nuclear4d2 1874 13386 4186 18470 8637
28651 Soybean seedling Flood + ABA treatment Nuclear4d3 1791 12789 4068 18286 8338
28652 Soybean seedling Flood + ABA treatment Nuclear2dFlood with ABA1 2057 15070 4585 19724 8709
28653 Soybean seedling Flood + ABA treatment Nuclear2dFlood with ABA2 1796 12799 3888 18513 7954
28654 Soybean seedling Flood + ABA treatment Nuclear2dFlood with ABA3 1753 11909 3703 18511 7593
28655 Soybean seedling Flood + ABA treatment Nuclear2dFlood1 2032 14726 4508 19366 8675
28656 Soybean seedling Flood + ABA treatment Nuclear2dFlood2 1623 11861 3532 18285 7595
28657 Soybean seedling Flood + ABA treatment Nuclear2dFlood3 1879 13119 3999 18655 8181
28658 Soybean seedling Flood + ABA treatment Total2d1 1858 15916 4308 20217 8875
28659 Soybean seedling Flood + ABA treatment Total2d2 1804 15692 4234 20428 8825
28660 Soybean seedling Flood + ABA treatment Total2d3 1796 15671 4325 20466 8922
28661 Soybean seedling Flood + ABA treatment Total4d1 1838 16188 4320 20251 8957
28662 Soybean seedling Flood + ABA treatment Total4d2 1774 14987 4135 19850 8459
28663 Soybean seedling Flood + ABA treatment Total4d3 1810 15520 4191 19867 8606
28664 Soybean seedling Flood + ABA treatment Total2dFlood with ABA1 1847 17525 4407 19975 9514
28665 Soybean seedling Flood + ABA treatment Total2dFlood with ABA2 1797 15852 4225 19770 9054
28666 Soybean seedling Flood + ABA treatment Total2dFlood with ABA3 1804 15778 4271 20127 8938
28667 Soybean seedling Flood + ABA treatment Total2dFlood1 1789 16229 4250 20183 8993
28668 Soybean seedling Flood + ABA treatment Total2dFlood2 1818 16461 4347 20219 9251
28669 Soybean seedling Flood + ABA treatment Total2dFlood3 1765 15949 4225 19747 9049

You are about to run PRIDE Inspector via Java Web Start. Double click on the JNLP file downloaded if it didn't start automatically.

Note: Due to Oracle's recent Java update, you may experience security related problems with Java Web Start. Please add "http://www.ebi.ac.uk" to the URLs listed in the Security tab of your Java Control Panel.