Project PXD000160

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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(s) : 23808807

Data Processing Protocol

See details in reference(s) : 23808807

Contact

Yohei Nanjo, Institute of Crop Science

Submission Date

26/02/2013

Publication Date

12/12/2013

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: 23808807

Assay

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Showing 1 - 10 of 24 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 28646 Soybean seedling Flood + ABA treatment Nuclear2d1 1519 13389 3296 18645 7660
2 28647 Soybean seedling Flood + ABA treatment Nuclear2d2 1818 14759 4103 19555 9051
3 28648 Soybean seedling Flood + ABA treatment Nuclear2d3 1848 14037 4282 19366 9108
4 28649 Soybean seedling Flood + ABA treatment Nuclear4d1 1661 12957 3710 18577 8091
5 28650 Soybean seedling Flood + ABA treatment Nuclear4d2 1874 13386 4186 18470 8637
6 28651 Soybean seedling Flood + ABA treatment Nuclear4d3 1791 12789 4068 18286 8338
7 28652 Soybean seedling Flood + ABA treatment Nuclear2dFlood with ABA1 2057 15070 4585 19724 8709
8 28653 Soybean seedling Flood + ABA treatment Nuclear2dFlood with ABA2 1796 12799 3888 18513 7954
9 28654 Soybean seedling Flood + ABA treatment Nuclear2dFlood with ABA3 1753 11909 3703 18511 7593
10 28655 Soybean seedling Flood + ABA treatment Nuclear2dFlood1 2032 14726 4508 19366 8675