Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis.
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.


CONCLUSIONS
The 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.
Plant Genetics Research Unit, USDA, Agricultural Research Service, 102 Curtis Hall, University of Missouri, Columbia, MO 65211 USA; Division of Biochemistry, University of Missouri, Columbia, MO 65211 USA. Electronic address: Jan.Miernyk@ars.usda.gov.
Ján A
JA
Miernyk
School of Social Work, University of Missouri, Columbia, MO 65211 USA.
Alissa A
AA
Jett
Plant Genetics Research Unit, USDA, Agricultural Research Service, 102 Curtis Hall, University of Missouri, Columbia, MO 65211 USA.
Mark L
ML
Johnston
D000107:Acetylation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005374:Filtration; D006031:Glycosylation; D008856:Microscopy, Fluorescence; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D030262:Soybean Proteins / Q000378:metabolism*; D013025:Glycine max / Q000254:growth & development / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D046509:Tissue Culture Techniques; D014357:Trypsin / Q000737:chemistry
D020543:Proteome; D030262:Soybean Proteins; D014357:Trypsin
DIGE; Glycine max; Protein dynamics; Secretome; Tandem mass spectrometry; Tryptic peptides