Project PXD000201

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Proteomic Change Associated with Flooding Injury of Soybean


Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. Two-day-old soybeans, seedlings flooded for 2 days and hypocotyls of seedlings grown for 3 days after flooding were analyzed. The radicles of soybean seedlings were separated to root tips (RT), roots without tip (RwoT) and hypocotyls (Hypo). Protein was extracted from these organs by TCA/Acetone method. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin. 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 6 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, 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 1.3, Thermo Fisher Scientific)

Sample Processing Protocol

See details in reference(s) : 23659366

Data Processing Protocol

See details in reference(s) : 23659366


Yohei Nanjo, Institute of Crop Science

Submission Date


Publication Date


Cell Type

plant cell


Not available

Experiment Type

Bottom-up proteomics

Assay count



    Nanjo Y, Nakamura T, Komatsu S; Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics., J Proteome Res, 2013 Nov 1, 12, 11, 4785-98, PubMed: 23659366


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 28967 4d-old Soybean root tip flooded for 2d with 120mL water 1 1119 6855 1992 17787 4321
2 28968 4d-old Soybean root tip flooded for 2d with 120mL water 1ii 1224 7156 2243 16379 4549
3 28969 4d-old Soybean root tip flooded for 2d with 120mL water 2 1111 7123 2038 17660 4377
4 28970 4d-old Soybean root tip flooded for 2d with 120mL water 2ii 1229 7505 2317 17449 4669
5 28971 4d-old Soybean root tip flooded for 2d with 120mL water 3 1154 7099 2090 17750 4379
6 28972 4d-old Soybean root tip flooded for 2d with 120mL water 3ii 1214 7172 2208 16312 4395
7 28973 2d-old Soybean root tip untreated 1 1064 5966 1843 16142 3754
8 28974 2d-old Soybean root tip untreated 1ii 1165 6216 2071 16025 3956
9 28975 2d-old Soybean root tip untreated 2 1080 6728 1925 17566 4102
10 28976 2d-old Soybean root tip untreated 2ii 1179 6590 2133 16100 4143