Submitted to: BARC Poster Day
Publication Type: Abstract only
Publication Acceptance Date: 4/1/2004
Publication Date: 4/1/2004
Citation: Natarajan, S.S., Xu, C., Caperna, T.J., Garrett, W.M. 2004. Proteome analysis of soybean proteins [abstract]. BARC Poster Day. Abstract p.17. Interpretive Summary:
Technical Abstract: Proteome analysis is an indispensable tool that can be used both to visualize and compare complex mixtures of proteins. These tools can also provide unique information about the individual proteins involved in specific biological processes. We have used two-dimensional (2-D) gel electrophoresis, Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of soybean seed proteins. In this study, we compared four different methods to solubilize proteins, including trichloroacetic acid (TCA)/acetone and phenol precipitation, and extraction in urea-lysis buffer with and without thiourea to determine the method that yielded the best-separated soybean seed proteins by 2D-PAGE. Gels were stained with colloidal Coomassie blue and scanned with a laser densitometer. Compared to the other methods, the modified TCA/acetone method showed higher protein resolution, and spot intensity than the other three methods. MALDI-TOF analysis of trypsinized protein spots cut from stained 2D gels showed several abundant proteins such as beta conglycinin with 3 subunits and glycinin with acidic and basic polypeptide chains. In addition, non-abundant proteins such as allergen Gly m Bd and seed maturation protein were identified by MALDI-TOF. Proteins which could not be positively identified by MALDI-TOF were further analyzed by LC-MS. Our results indicate that the combination of MALDI-TOF and LC-MS analysis is an effective method for the identification of both abundant and non-abundant soybean seed proteins.