Project : PXD000247

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Summary

Title

Proteomic analysis of the testa from developing soybean seeds

Description

Soybean (Glycine max (L.) Merr. cv Jack) seed development was separated into nine defined stages (S1 to S9). Testa (seed coats) were removed from developing seeds at stages S2, 4, 6, 8, and 9, and subjected to shotgun proteomic profiling. For each stage "total proteins" were isolated from 150mg dry weight of seed coat using a phenol-based method, then reduced, alkylated, and digested with trypsin. The tryptic peptides were separated using a C18-reversed phase matrix, then analyzed using an LTQ Orbitrap Mass Spectrometer. Spectra were searched against the Phytozome G. max DB using the Sorcerer 2 IDA Sequest-based search algorithm. Identities were verified using Scaffold 3. A total of 306 (S2), 328 (S4), 273 (S6), 193 (S8), and 272 (S9) proteins were identified in three out of three biological replicates, and sorted into 11 functional groups: Primary Metabolism, Secondary Metabolism, Cellular Structure, Stress Responses, Nucleic Acid metabolism, Protein Synthesis, Protein Folding, Protein Targeting, Hormones and Signaling, Seed Storage Proteins, and Proteins of Unknown Function. In selected instances, individual seed coat proteins were quantified by spectral counting. The number of proteins involved in intermediary metabolism, flavonoid biosynthesis, protein folding and degradation are discussed as they relate to seed coat function. A database was constructed using the open reading frames of Phytozome G. max DB (updated August 8, 2009), plus a decoy DB generated by reversing the target sequences. Spectra were searched against the combined target-decoy DB using the Sorcerer 2 IDA Sequest-based search algorithm. Identities were verified using the Scaffold 3 program, and the false discovery rate (FDR) was estimated as (Ndecoy/Ntarget). For 186,364 peptide spectra examined, the FDR was between 5.18 and 5.26%. Samples were analyzed in biological triplicate; proteins were included in the results only if they were identified in all three biological replicates. Relative protein abundance was quantified by spectral counting ; data presented are the mean of three independent determinations. The coefficient of variation values calculated for each developmental stage are < 0.0516.

Sample Processing Protocol

See details in reference PMID : 23707235

Data Processing Protocol

See details in reference PMID : 23707235

Contact

Jan Miernyk, USDA Agriculture Research Service

Submission Date

08/05/2013

Publication

    Miernyk JA, Johnston ML; Proteomic analysis of the testa from developing soybean seeds., J Proteomics, 2013 Aug 26, 89, 265-72, PubMed(s) : 23707235

Assay

Accession Title Protein Count Peptide Count Unique Peptide Count Spectrum Count Identified Spectrum Count
29517 Stage 2 soybean seed coat, replicate 1 2671 7676 2987 3446 3446
29518 Stage 2 soybean seed coat, replicate 2 2840 8297 3314 3809 3809
29519 Stage 2 soybean seed coat, replicate 3 2693 8124 3161 3681 3681
29520 Stage 6 soybean seed coat, replicate 1 2846 8209 3378 3899 3899
29521 Stage 6 soybean seed coat, replicate 2 2722 8916 3638 4229 4229
29522 Stage 6 soybean seed coat, replicate 3 2561 6871 2785 3248 3248
29523 Stage 8 soybean seed coat, replicate 1 2315 6866 3001 3635 3635
29524 Stage 8 soybean seed coat, replicate 2 2182 5637 2487 2890 2890
29525 Stage 8 soybean seed coat, replicate 3 2146 5623 2519 2919 2919
29526 Stage 4 soybean seed coat, replicate 1 2916 10501 4024 4759 4759
29527 Stage 4 soybean seed coat, replicate 2 3044 10475 3825 4516 4516
29528 Stage 4 soybean seed coat, replicate 3 3022 10522 3980 4582 4582
29529 Stage 9 soybean seed coat, replicate 1 2256 7591 2978 4116 4116
29530 Stage 9 soybean seed coat, replicate 2 2124 7239 2941 3978 3978
29531 Stage 9 soybean seed coat, replicate 3 2143 7165 2933 3953 3953

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