Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

• Difference gel electrophoresis was used to study protein dynamics during the culture cycle of soybean suspension cells.
• The extracellular proteome (secretome) is both dynamic and distinct from the intracellular proteome.
• There were no indications of N-glycosylation, O-phosphorylation, of K-acetylation of extracellular proteins.
• The bases for extracellular protein dynamics likely involve both proteolytic and redox modifications.

Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transferred to fresh medium at 7-day intervals. Cultures were harvested by filtration five days (early log phase) or eight days (late log phase) after transfer. In order to evaluate dynamic changes, both intracellular and extracellular proteins were analyzed by 2-dimensional difference gel electrophoresis. Selected spots were subjected to in-gel tryptic-digestion and the resultant peptides were analyzed by nLC-MS/MS. In follow-up studies gel-free shot-gun analyses led to identification of 367 intracellular proteins and 188 extracellular proteins.SignificanceThe significance of the described research is two-fold. First a gel-based proteomics method was applied to the study of the dynamics of the secretome (extracellular proteins). Second, results of a shot-gun non-gel based proteomic survey of both cellular and extracellular proteins are presented.

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