Proteomic analysis of the testa from developing soybean seeds.

UNLABELLED 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, 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. BIOLOGICAL SIGNIFICANCE Most previous analyses of seed coats have either targeted individual enzymes or used the results from high-throughput transcript profiling to infer biological function. Because there is seldom a linear correlation between transcript and protein levels, we have undertaken a shotgun proteomics-based description of soybean (G. max (L.) Merr. cv Jack) seed coats, as a function of development, in order to bridge this gap and to establish the baseline for a more comprehensive understanding of seed biology.