Proteomic analysis of the testa from developing soybean seeds
2013
Miernyk, Jan A. | Johnston, Mark L.
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 150 mg 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 with a continuous gradient of acetonitrile in 0.1% formic acid, then analyzed using an LTQ Orbitrap Mass Spectrometer. Following a high-resolution FTMS scan of the eluting peptides, each second the 9 most abundant peptides were fragmented by CID in the ion-trap. 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 451 (S2), 558 (S4), 455 (S6), 347 (S8), and 409 (S9) proteins were identified, 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 abundance of proteins involved in intermediary metabolism, flavonoid biosynthesis, protein folding and degradation are discussed as they relate to seed coat function.
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